FXR (NR1H4) modulating compounds

ABSTRACT

The present disclosure relates generally to compounds which bind to the NR1H4 receptor (FXR) and act as agonists of FXR. The disclosure further relates to the use of the compounds for the preparation of a medicament for the treatment of diseases and/or conditions through binding of said nuclear receptor by said compounds and to a process for the synthesis of said compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/541,073, filed Aug. 14, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/618,666, filed Jun. 9, 2017, now U.S. Pat. No.10,421,730, which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/349,490, filed Jun. 13, 2016, all ofwhich are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 1165_PC_ST25.txt. The text file created on May16, 2017, is about 550 bytes and submitted electronically via EFS-Web.

FIELD

The present disclosure relates to compounds which bind to the NR1H4receptor (FXR) and act as agonists or modulators of FXR. The disclosurefurther relates to the use of the compounds for the treatment and/orprophylaxis of diseases and/or conditions through binding of saidnuclear receptor by said compounds.

BACKGROUND

Multicellular organisms are dependent on advanced mechanisms ofinformation transfer between cells and body compartments. Theinformation that is transmitted can be highly complex and can result inthe alteration of genetic programs involved in cellular differentiation,proliferation, or reproduction. The signals, or hormones, are often lowmolecular weight molecules, such as peptides, fatty acid, or cholesterolderivatives.

Many of these signals produce their effects by ultimately changing thetranscription of specific genes. One well-studied group of proteins thatmediate a cell's response to a variety of signals is the family oftranscription factors known as nuclear receptors, hereinafter referredto often as “NR.” Members of this group include receptors for steroidhormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroidhormone, bile acids, cholesterol-derivatives, fatty acids (and otherperoxisomal proliferators), as well as so-called orphan receptors,proteins that are structurally similar to other members of this group,but for which no ligands are known. Orphan receptors may be indicativeof unknown signalling pathways in the cell or may be nuclear receptorsthat function without ligand activation. The activation of transcriptionby some of these orphan receptors may occur in the absence of anexogenous ligand and/or through signal transduction pathways originatingfrom the cell surface.

In general, three functional domains have been defined in NRs. An aminoterminal domain is believed to have some regulatory function. It isfollowed by a DNA-binding domain (hereinafter referred to as “DBD”),which usually comprises two zinc finger elements and recognizes aspecific Hormone Responsive Element (hereinafter referred to as “HRE”)within the promoters of responsive genes. Specific amino acid residuesin the “DBD” have been shown to confer DNA sequence binding specificity.A ligand-binding-domain (hereinafter referred to as “LBD”) is at thecarboxy-terminal region of known NRs.

In the absence of hormone, the LBD appears to interfere with theinteraction of the DBD with its HRE. Hormone binding seems to result ina conformational change in the NR and thus opens this interference. A NRwithout the LBD constitutively activates transcription but at a lowlevel.

Coactivators or transcriptional activators are proposed to bridgebetween sequence specific transcription factors, and the basaltranscription machinery and in addition to influence the chromatinstructure of a target cell. Several proteins like SRC-1, ACTR, and Griplinteract with NRs in a ligand enhanced manner.

Nuclear receptor modulators like steroid hormones affect the growth andfunction of specific cells by binding to intracellular receptors andforming nuclear receptor-ligand complexes. Nuclear receptor-hormonecomplexes then interact with a HRE in the control region of specificgenes and alter specific gene expression.

The Farnesoid X Receptor alpha (hereinafter also often referred to asNR1H4 when referring to the human receptor) is a prototypical type 2nuclear receptor which activates genes upon binding to a promoter regionof target genes in a heterodimeric fashion with Retinoid X Receptor. Therelevant physiological ligands of NR1H4 are bile acids. The most potentone is chenodeoxycholic acid (CDCA), which regulates the expression ofseveral genes that participate in bile acid homeostasis. Farnesol andderivatives, together called farnesoids, are originally described toactivate the rat orthologue at high concentration but they do notactivate the human or mouse receptor. FXR is expressed in the liver,throughout the entire gastrointestinal tract including the esophagus,stomach, duodenum, small intestine, colon, ovary, adrenal gland andkidney. Beyond controlling intracellular gene expression, FXR seems tobe also involved in paracrine and endocrine signalling by upregulatingthe expression of the cytokine Fibroblast Growth Factor 15 (rodents) or19 (monkeys, humans A).

Although numerous FXR agonists are known, there is a need for improvedFXR agonists.

SUMMARY

The present disclosure provides compounds bind to the NR1H4 receptor(FXR) and act as agonists or modulators of FXR. The disclosure furtherrelates to the use of the compounds for the treatment and/or prophylaxisof diseases and/or conditions through binding of said nuclear receptorby said compounds.

The present disclosure provides compounds according to Formula (I):

wherein:

Q is phenylene or pyridylene, each of which is optionally substitutedwith one or two substituents independently selected from halogen,methyl, C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy, —CH₂F, —CHF₂, and —CF₃;

Y is N or CH;

A is pyridylene or phenylene, each of which is optionally substitutedwith one or two groups independently selected from halogen, C₁₋₄-alkoxy,halo-C₁₋₄-alkoxy, C₁₋₄-alkyl, and halo-C₁₋₄-alkyl;

Z is isoxazole substituted with R¹ or pyrazole substituted with R¹;

R¹ is C₁₋₄-alkyl or C₃₋₆-cycloalkyl, wherein

-   -   said C₁₋₄-alkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkoxy, and fluoro-C₁₋₃-alkoxy, and    -   said C₃₋₆-cycloalkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkyl, fluoro-C₁₋₃-alkyl, C₁₋₃-alkoxy, and        fluoro-C₁₋₃-alkoxy;

R² and R³ are independently selected from hydrogen, halogen, methoxy,—CF₃, —CHF₂, —CH₂F, —OCH₂F, —OCHF₂, —OCF₃, and methyl;

-   -   R⁴ is —CO₂R⁵ or —C(O)NR⁵R⁶;    -   R⁵ is hydrogen, C₁₋₆-alkyl, or halo-C₁₋₆-alkyl; and    -   R⁶ is hydrogen or C₁₋₆-alkyl, wherein said C₁₋₆-alkyl is        optionally substituted with 1 to 6 substituents independently        selected from halogen, —SO₃H, and —CO₂H;        or a pharmaceutically acceptable salt, a stereoisomer, a mixture        of stereoisomers, or a tautomer thereof.

Some embodiments provide for pharmaceutical compositions comprising acompound of formula (I) and a pharmaceutically acceptable excipient.

Also provided herein are methods of treating a patient having an FXRmediated condition comprising administering a compound of formula (I) toa patient in need thereof.

DESCRIPTION OF THE FIGURES

FIG. 1: Plasma exposure of Example 3 and Comparative Example 2 versusplasma FGF19 levels in cynomolgus monkey.

FIG. 2: FGF19 levels generated in cynomolgus monkey with increasing oraldoses of Example 3 and Comparative Example 2.

DETAILED DESCRIPTION Definitions

The following description sets forth exemplary embodiments of thepresent technology. It should be recognized, however, that suchdescription is not intended as a limitation on the scope of the presentdisclosure but is instead provided as a description of exemplaryembodiments.

As used in the present specification, the following words, phrases andsymbols are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise.

The disclosures illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising”, “including,” “containing”, etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —C(O)NH₂is attached through the carbon atom. A dash at the front or end of achemical group is a matter of convenience; chemical groups may bedepicted with or without one or more dashes without losing theirordinary meaning. A wavy line drawn through a line in a structureindicates a point of attachment of a group. Unless chemically orstructurally required, no directionality is indicated or implied by theorder in which a chemical group is written or named.

The prefix “C_(u-v)” indicates that the following group has from u to vcarbon atoms. For example, “C₁₋₆ alkyl” indicates that the alkyl grouphas from 1 to 6 carbon atoms.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. In certain embodiments, the term “about” includes the indicatedamount ±10%. In other embodiments, the term “about” includes theindicated amount ±5%. In certain other embodiments, the term “about”includes the indicated amount ±1%. Also, to the term “about X” includesdescription of “X”. Also, the singular forms “a” and “the” includeplural references unless the context clearly dictates otherwise. Thus,e.g., reference to “the compound” includes a plurality of such compoundsand reference to “the assay” includes reference to one or more assaysand equivalents thereof known to those skilled in the art.

In the context of the present disclosure “alkyl” means a saturatedhydrocarbon chain, which may be straight chained or branched. In thecontext of the present disclosure, “C₁₋₆-alkyl” means a saturated alkylchain having 1 to 6 carbon atoms which may be straight chained orbranched. Examples thereof include methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl andn-hexyl.

The term “haloalkyl” means that one or more hydrogen atoms in the alkylchain are replaced by a halogen. A non-limiting example thereof is CF₃.

A “cycloalkyl” group means a saturated or partially unsaturated mono-,bi- or spirocyclic hydrocarbon ring system.

An “alkoxy” group refers to —O-alkyl, wherein alkyl is as definedherein. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and1,2-dimethylbutoxy.

“Halogen” or “halo” refers to a F, Cl, Br, or I atom.

“Hydroxyl” or “hydroxy” refers to —OH.

“Haloalkoxy” refers to an alkoxy group as defined herein wherein one ormore hydrogen atoms in the alkyl chain are replaced by a halogen.

“Fluoroalkyl” refers to an alkyl group as defined herein wherein one ormore hydrogen atoms in the alkyl chain are replaced by fluoro.

“Fluoroalkoxy” refers to an alkoxy group as defined herein wherein oneor more hydrogen atoms in the alkyl chain are replaced by fluoro.

The terms “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. Also, the term “optionallysubstituted” refers to any one or more hydrogen atoms on the designatedatom or group may or may not be replaced by a moiety other thanhydrogen.

Furthermore, the compounds of the present disclosure may be subject totautomerism. Where tautomerism, e.g. keto-enol tautomerism, of compoundsof the present disclosure or their prodrugs may occur, the individualforms, like e.g. the keto and enol form, are each within the scope ofthe disclosure as well as their mixtures in any ratio. The same appliesfor stereoisomers, like e.g. enantiomers, cis/trans isomers, conformersand the like.

The term “protecting group” refers to a moiety of a compound that masksor alters the properties of a functional group or the properties of thecompound as a whole. Chemical protecting groups and strategies forprotection/deprotection are well known in the art. See e.g., ProtectiveGroups in Organic Chemistry, Theodora W. Greene, John Wiley & Sons,Inc., New York, 1991. Protecting groups are often utilized to mask thereactivity of certain functional groups, to assist in the efficiency ofdesired chemical reactions, e.g., making and breaking chemical bonds inan ordered and planned fashion. The term “deprotecting” refers toremoving the protecting group.

A “leaving group” includes a molecular fragment that can depart with apair of electrons from a covalent bond to the reacting carbon atomduring a chemical reaction.

It will be appreciated by the skilled person that when lists ofalternative substituents include members which, because of their valencyrequirements or other reasons, cannot be used to substitute a particulargroup, the list is intended to be read with the knowledge of the skilledperson to include only those members of the list which are suitable forsubstituting the particular group.

In some embodiments, the compounds of the present disclosure can be inthe form of a “prodrug.” The term “prodrug” is defined in thepharmaceutical field as a biologically inactive derivative of a drugthat upon administration to the human body is converted to thebiologically active parent drug according to some chemical or enzymaticpathway. Examples of prodrugs include esterified carboxylic acids.

In the human liver, UDP-glucuronosyltransferases act on certaincompounds having amino, carbamyl, thio (sulfhydryl) or hydroxyl groupsto conjugate uridine diphosphate-α-D-glucuronic acid through glycosidebonds, or to esterify compounds with carboxy or hydroxyl groups in theprocess of phase II metabolism. Compounds of the present disclosure maybe glucuronidated, that is to say, conjugated to glucuronic acid, toform glucuronides, particularly ((3-D)glucuronides.

One step in the formation of bile is the conjugation of the individualbile acids with an amino acid, particularly glycine or taurine.Compounds of the present disclosure may be conjugated with glycine ortaurine at a substitutable position.

The compounds of the present disclosure can be in the form of apharmaceutically acceptable salt. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases or acids, including inorganic bases or acids and organicbases or acids. In case the compounds of the present disclosure containone or more acidic or basic groups, the disclosure also comprises theircorresponding pharmaceutically or toxicologically acceptable salts, inparticular their pharmaceutically utilizable salts. Thus, the compoundsof the present disclosure which contain acidic groups can be present onthese groups and can be used according to the disclosure, for example,as alkali metal salts, alkaline earth metal salts or ammonium salts.More precise examples of such salts include sodium salts, potassiumsalts, calcium salts, magnesium salts or salts with ammonia or organicamines such as, for example, ethylamine, ethanolamine, triethanolamineor amino acids. The compounds of the present disclosure which containone or more basic groups, i.e. groups which can be protonated, can bepresent and can be used according to the disclosure in the form of theiraddition salts with inorganic or organic acids. Examples of suitableacids include hydrogen chloride, hydrogen bromide, phosphoric acid,sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonicacid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaricacid, lactic acid, salicylic acid, benzoic acid, formic acid, propionicacid, pivalic acid, diethylacetic acid, malonic acid, succinic acid,pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid,phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid,citric acid, adipic acid, and other acids known to the person skilled inthe art. If the compounds of the present disclosure simultaneouslycontain acidic and basic groups in the molecule, the disclosure alsoincludes, in addition to the salt forms mentioned, inner salts orbetaines (zwitterions). The respective salts can be obtained bycustomary methods which are known to the person skilled in the art like,for example, by contacting these with an organic or inorganic acid orbase in a solvent or dispersant, or by anion exchange or cation exchangewith other salts. The present disclosure also includes all salts of thecompounds of the present disclosure which, owing to low physiologicalcompatibility, are not directly suitable for use in pharmaceuticals butwhich can be used, for example, as intermediates for chemical reactionsor for the preparation of pharmaceutically acceptable salts.

Further the compounds of the present disclosure may be present in theform of solvates, such as those which include as solvate water, orpharmaceutically acceptable solvates, such as alcohols, in particularethanol. A “solvate” is formed by the interaction of a solvent and acompound.

In certain embodiments, provided are optical isomers, racemates, orother mixtures thereof of the compounds described herein or apharmaceutically acceptable salt or a mixture thereof. If desired,isomers can be separated by methods well known in the art, e.g. byliquid chromatography. In those situations, the single enantiomer ordiastereomer, i.e., optically active form, can be obtained by asymmetricsynthesis or by resolution. Resolution can be accomplished, for example,by conventional methods such as crystallization in the presence of aresolving agent, or chromatography, using for example, a chiral highpressure liquid chromatography (HPLC) column.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers,”which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another. “Diastereomers” are stereoisomers thathave at least two asymmetric atoms, but which are not mirror-images ofeach other.

The compounds disclosed herein and their pharmaceutically acceptablesalts may include an asymmetric center and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centres of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers.

Compositions provided herein that include a compound described herein orpharmaceutically acceptable salts, isomer, or a mixture thereof mayinclude racemic mixtures, or mixtures containing an enantiomeric excessof one enantiomer or single diastereomers or diastereomeric mixtures.All such isomeric forms of these compounds are expressly included hereinthe same as if each and every isomeric form were specifically andindividually listed.

Any formula or structure given herein, is also intended to representunlabeled forms as well as isotopically labeled forms of the compounds.Isotopically labeled compounds have structures depicted by the formulasgiven herein except that one or more atoms are replaced by an atomhaving a selected atomic mass or mass number. Examples of isotopes thatcan be incorporated into compounds of the disclosure include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as, but not limited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C,¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶Cl and ¹²⁵I. Various isotopicallylabeled compounds of the present disclosure, for example those intowhich radioactive isotopes such as ³H, ¹³C and ¹⁴C are incorporated.Such isotopically labelled compounds may be useful in metabolic studies,reaction kinetic studies, detection or imaging techniques, such aspositron emission tomography (PET) or single-photon emission computedtomography (SPECT) including drug or substrate tissue distributionassays or in radioactive treatment of patients. Isotopically labeledcompounds of this disclosure and prodrugs thereof can generally beprepared by carrying out the procedures disclosed in the schemes or inthe examples and preparations described below by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

The disclosure also includes “deuterated analogs” of compounds ofFormula (I) in which from 1 to n hydrogens attached to a carbon atomis/are replaced by deuterium, in which n is the number of hydrogens inthe molecule. Such compounds may exhibit increased resistance tometabolism and thus be useful for increasing the half-life of anycompound of Formula I when administered to a mammal, e.g. a human. See,for example, Foster, “Deuterium Isotope Effects in Studies of DrugMetabolism,” Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compoundsare synthesized by means well known in the art, for example by employingstarting materials in which one or more hydrogens have been replaced bydeuterium.

Deuterium labelled or substituted therapeutic compounds of thedisclosure may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life, reduced dosage requirements and/oran improvement in therapeutic index. An ¹⁸F labeled compound may beuseful for PET or SPECT studies.

The concentration of such a heavier isotope, specifically deuterium, maybe defined by an isotopic enrichment factor. In the compounds of thisdisclosure any atom not specifically designated as a particular isotopeis meant to represent any stable isotope of that atom. Unless otherwisestated, when a position is designated specifically as “H” or “hydrogen”,the position is understood to have hydrogen at its natural abundanceisotopic composition. Accordingly, in the compounds of this disclosureany atom specifically designated as a deuterium (D) is meant torepresent deuterium.

Furthermore, the present disclosure provides pharmaceutical compositionscomprising at least one compound of the present disclosure, or a prodrugcompound thereof, or a pharmaceutically acceptable salt or solvatethereof as active ingredient together with a pharmaceutically acceptablecarrier.

“Pharmaceutical composition” means one or more active ingredients, andone or more inert ingredients that make up the carrier, as well as anyproduct which results, directly or indirectly, from combination,complexation or aggregation of any two or more of the ingredients, orfrom dissociation of one or more of the ingredients, or from other typesof reactions or interactions of one or more of the ingredients.Accordingly, the pharmaceutical compositions of the present disclosureencompass any composition made by admixing at least one compound of thepresent disclosure and a pharmaceutically acceptable carrier.

List of Abbreviations and Acronyms Abbreviation Meaning (±)-BINAP(±)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene 2-MeTHF 2-methyltetrahydrofuran ACN or MeCN Acetonitrile aq. aqueous Bn Benzyl BOC orBoc t-Butyloxycarbonyl BSA Bovine serum albumin BSS Balanced SaltSolution calcd calculated DAST (diethylamino)sulfur trifluoride DCMDichloromethane DIBAL-H Diisobutylaluminum hydride DMF DimethylformamideDMSO Dimethylsulfoxide EA Ethyl acetate EDTA Ethylenediaminetetraaceticacid ESI Electronspray Ionization Et Ethyl Et₂O Diethyl ether EtOAcEthyl acetate FBS Fetal bovine serum h or hr(s) Hour(s) HATU1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate HPLC High performance liquid chromatographyIPA Isopropyl alcohol IPTG Isopropyl β-D-1-thiogalactopyranoside LCMS orLiquid Chromatography Mass Spectrometry LC/MS Me Methyl MEM MinimumEssential Medium MeOH Methanol min Minute(s) MS Mass Spectrometry m/zMass-to-charge ratio NADPH Dihydronicotinamide-adenine dinucleotidephosphate NMP N-methylpyrrolidone NMR Nuclear Magnetic Resonancespectroscopy n-BuLi n-butyllithium rpm Revolutions per minute PEPetroleum ether RT or rt Room temperature sat. saturated TBAFTetrabutylammonium fluoride TBDMS t-butyldimethylsilyl TBSt-butyldimethylsilyl TEMPO 2,2,6,6-Tetramethylpiperidine 1-oxyl TFATrifluoroacetic acid THF tetrahydrofuran TMS trimethylsilyl UPLC UltraPerformance Liquid Chromatography

Compounds

Provided herein are compounds according to Formula (I):

wherein:

Q is phenylene or pyridylene, each of which is optionally substitutedwith one or two substituents independently selected from halogen,methyl, C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy, —CH₂F, —CHF₂, and —CF₃;

Y is N or CH;

A is pyridylene or phenylene, each of which is optionally substitutedwith one or two groups independently selected from halogen, C₁₋₄-alkoxy,halo-C₁₋₄-alkoxy, C₁₋₄-alkyl, and halo-C₁₋₄-alkyl;

Z is isoxazole substituted with R¹ or pyrazole substituted with R¹;

R¹ is C₁₋₄-alkyl or C₃₋₆-cycloalkyl, wherein

-   -   said C₁₋₄-alkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkoxy, and fluoro-C₁₋₃-alkoxy, and    -   said C₃₋₆-cycloalkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkyl, fluoro-C₁₋₃-alkyl, C₁₋₃-alkoxy, and        fluoro-C₁₋₃-alkoxy;

R² and R³ are independently selected from hydrogen, halogen, methoxy,—CF₃, —CHF₂, —CH₂F, —OCH₂F, —OCHF₂, —OCF₃, and methyl;

R⁴ is —CO₂R⁵ or —C(O)NR⁵R⁶;

R⁵ is hydrogen, C₁₋₆-alkyl, or halo-C₁₋₆-alkyl; and

R⁶ is hydrogen or C₁₋₆-alkyl, wherein said C₁₋₆-alkyl is optionallysubstituted with 1 to 6 substituents independently selected fromhalogen, —SO₃H, and —CO₂H;

or a pharmaceutically acceptable salt, a stereoisomer, a mixture ofstereoisomers, or a tautomer thereof.

One embodiment provides for compounds of Formula (Ia):

wherein:

Q is phenylene or pyridylene, each of which is optionally substitutedwith one or two substituents independently selected from halogen,methyl, C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy, —CH₂F, —CHF₂, and —CF₃;

Y is N or CH;

A is pyridylene or phenylene, each of which is optionally substitutedwith one or two groups independently selected from halogen, C₁₋₄-alkoxy,halo-C₁₋₄-alkoxy, C₁₋₄-alkyl, and halo-C₁₋₄-alkyl;

R¹ is C₁₋₄-alkyl or C₃₋₆-cycloalkyl, wherein

-   -   said C₁₋₄-alkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkoxy, and fluoro-C₁₋₃-alkoxy, and    -   said C₃₋₆-cycloalkyl is optionally substituted with 1 to 3        substituents independently selected from fluoro, hydroxyl,        C₁₋₃-alkyl, fluoro-C₁₋₃-alkyl, C₁₋₃-alkoxy, and        fluoro-C₁₋₃-alkoxy;

R² and R³ are independently selected from hydrogen, halogen, methoxy,—CF₃, —CHF₂, —CH₂F, —OCH₂F, —OCHF₂, —OCF₃, and methyl;

R⁴ is —CO₂R⁵ or —C(O)NR⁵R⁶;

R⁵ is hydrogen, C₁₋₆-alkyl, or halo-C₁₋₆-alkyl;

R⁶ is hydrogen or C₁₋₆-alkyl, wherein said C₁₋₆-alkyl is optionallysubstituted with 1 to 6 substituents independently selected fromhalogen, —SO₃H, and —CO₂H;

or a pharmaceutically acceptable salt, a stereoisomer, a mixture ofstereoisomers, or a tautomer thereof.

One embodiment provides for compounds of formula (Ia):

wherein:

-   -   Q is phenylene optionally substituted with one or two halogen;    -   Y is N or CH;    -   A is pyridylene optionally substituted with one or two groups        independently selected from halogen and C₁₋₄-alkoxy;    -   R¹ is C₁₋₄-alkyl or C₃₋₆-cycloalkyl;    -   R² and R³ are independently selected from hydrogen and halogen;    -   R⁴ is —CO₂R⁵ or —C(O)NR⁵R⁶;    -   R⁵ is hydrogen; and    -   R⁶ is C₁₋₂-alkyl optionally substituted with —CO₂H or —SO₃H;        or pharmaceutically acceptable salt, a stereoisomer, a mixture        of stereoisomers, or a tautomer thereof.

In one embodiment, Q is phenylene or pyridylene, each of which isoptionally substituted with one or two substituents independentlyselected from halogen, methyl, —CHF₂, and —CF₃. In some embodiments, Qis phenylene optionally substituted with one or two substituentsindependently selected from halogen, methyl, and —CF₃. In someembodiments, Q is pyridylene optionally substituted with one or twosubstituents independently selected from halogen, methyl, and —CF₃.

In one embodiment, Q is phenylene optionally substituted with one or twohalogen. In some embodiments, Q is pyridylene optionally substitutedwith one or two halogen. In some embodiments, Q is phenylene optionallysubstituted with one or two chloro. In some embodiments, Q is pyridyleneoptionally substituted with one or two chloro.

In one embodiment, Q is phenylene substituted with one chloro. In someembodiments, Q is pyridylene substituted with one chloro.

In one embodiment, R¹ is C₁₋₄-alkyl. In some embodiments, R¹ isC₃₋₆-cycloalkyl. In some embodiments, R¹ is cyclopropyl or methyl. Insome embodiments, R¹ is cyclopropyl.

In one embodiment, R² and R³ are not both hydrogen. In some embodiments,R² and R³ are independently selected from hydrogen, halogen, methoxy,—OCHF₂, —OCF₃, and methyl. In some embodiments, R² and R³ areindependently selected from halogen, methoxy, —OCHF₂, —OCF₃, and methyl.

In one embodiment, R² and R³ are halogen. In some embodiments, R² and R³are chloro.

In one embodiment, one of R² and R³ is a halogen and the other ishydrogen. In one embodiment, one of R² and R³ is a chloro and the otheris hydrogen. In some embodiments, one of R² and R³ is a fluoro and theother is hydrogen.

In one embodiment, Y is N. In some embodiments, Y is CH.

In one embodiment, A is pyridylene optionally substituted with one ortwo halogen. In some embodiments, A is pyridylene optionally substitutedwith one or two C₁₋₄-alkoxy.

In one embodiment, A is pyridylene substituted with one fluoro. In someembodiments, A is pyridylene substituted with one methoxy. In oneembodiment, A is unsubstituted pyridylene.

In one embodiment, A is phenylene optionally substituted with one or twohalogen. In one embodiment, A is phenylene optionally substituted withone or two C₁₋₄-alkoxy.

In one embodiment, A is phenylene substituted with one fluoro. In oneembodiment, A is phenylene substituted with one methoxy. In oneembodiment, A is unsubstituted phenylene.

In one embodiment, R⁴ is —CO₂R⁵, and R⁵ is hydrogen. In one embodiment,R⁴ is —CO₂R⁵ and R⁵ is C₁₋₆-alkyl or halo-C₁₋₆-alkyl.

In one embodiment, R⁴ is —C(O)NR⁵R⁶, R⁵ is C₁₋₆-alkyl orhalo-C₁₋₆-alkyl, and R⁶ is C₁₋₂-alkyl, wherein said C₁₋₂-alkyl issubstituted with —SO₃H or —CO₂H.

In one embodiment, R⁴ is —C(O)NR⁵R⁶, R⁵ is hydrogen, and R⁶ isC₁₋₂-alkyl, wherein said C₁₋₂-alkyl is substituted with —SO₃H or —CO₂H.

In one embodiment, R⁴-A is:

wherein the pyridylene is optionally substituted with one or two groupsindependently selected from halogen, C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy,C₁₋₄-alkyl, and halo-C₁₋₄-alkyl.

In one embodiment, R⁴-A is:

In one embodiment, R⁴-A is:

In one embodiment, R⁴-A is:

In one embodiment, R⁴-A is

In one embodiment, provided is a compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt, a stereoisomer, a mixture ofstereoisomers, or a tautomer thereof.

In one embodiment, provided herein is a compound having the followingformula:

or a pharmaceutically acceptable salt thereof.

In one embodiment, provided herein is a compound having the followingformula:

The chemical name of each of these compounds is provided in Table 1below.

TABLE 1 Example Structure IUPAC Name 1

2-(3-(2-chloro-4-((5- cyclopropyl-3-(2,4- difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1- yl)isonicotinic acid 2

2-(3-(2-chloro-4-((5- cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4- yl)methoxy)phenyl)-3- hydroxyazetidin-1-yl)isonicotinic acid 3

6-(3-(2-chloro-4-((5- cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4- yl)methoxy)phenyl)-3- hydroxyazetidin-1-yl)-5-fluoronicotinic acid 4

6-(3-(2-chloro-4-((3-(2,6- dichloro-4-fluorophenyl)-5- methylisoxazol-4-yl)methoxy)phenyl)-3- hydroxyazetidin-1-yl)-5- fluoronicotinic acid 5

6-(3-(2-chloro-4-((4- cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5- yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5- fluoronicotinic acid 6

5-((1S,3S)-3-(2-chloro-4-((5- cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4- yl)methoxy)phenyl)-3- hydroxycyclobutyl)-6-methoxynicotinic acid 7

2-(6-(3-(2-chloro-4-((5- cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4- yl)methoxy)phenyl)-3- hydroxyazetidin-1-yl)-5-fluoronicotinamido)ethane-1- sulfonic acid 8

(6-(3-(2-chloro-4-((5- cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4- yl)methoxy)phenyl-3- hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycine

Pharmaceutical Compositions and Modes of Administration

The present disclosure further provides pharmaceutical compositionscomprising at least one compound of the present disclosure, or aprodrug, a pharmaceutically acceptable salt, or solvate thereof asactive ingredient together with a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present disclosure mayadditionally comprise one or more other compounds as active ingredientslike a prodrug or other nuclear receptor modulators.

The compositions are suitable for oral, rectal, topical, parenteral(including subcutaneous, intramuscular, and intravenous), ocular(ophthalmic), pulmonary (nasal or buccal inhalation) or nasaladministration, although the most suitable route in any given case willdepend on the nature and severity of the conditions being treated and onthe nature of the active ingredient. They may be conveniently presentedin unit dosage form and prepared by any of the methods well-known in theart of pharmacy.

In practical use, the compounds of the present disclosure can becombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral (including intravenous). In preparing thecompositions for oral dosage form, any of the usual pharmaceutical mediamay be employed, such as, for example, water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like in thecase of oral liquid preparations, such as, for example, suspensions,elixirs and solutions; or carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents and the like in the case of oral solidpreparations such as, for example, powders, hard and soft capsules andtablets, with the solid oral preparations being preferred over theliquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are employed. If desired, tablets may be coatedby standard aqueous or non-aqueous techniques. Such compositions andpreparations should contain at least 0.1 percent of active compound. Thepercentage of active compound in these compositions may, of course, bevaried and may conveniently be between about 2 percent to about 60percent of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that an effective dosagewill be obtained. The active compounds can also be administeredintranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Since the compounds of the present disclosure mostly representcarboxylic acids or similar anionic isosters thereof, and since saltforms of ionic compounds can substantially affect bioavailability, thecompounds of the present disclosure may also be used as salts withvarious countercations to yield an orally available formulation. Suchpharmaceutically acceptable cations may be amongst others mono- orbivalent ions such as ammonium, the alkaline metals sodium or potassiumor the alkaline earth metals magnesium or calcium, certainpharmaceutically acceptable amines such astris(hydroxymethyl)aminomethane, ethylendiamine, diethylamine,piperazine or others, or certain cationic amino acids such as lysine orarginine.

The compounds of the present disclosure may also be administeredparenterally. Solutions or suspensions of these active compounds can beprepared in water suitably mixed with a surfactant such ashydroxy-propylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dose of a compound of thepresent disclosure. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, and the like may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, and the like. In some embodiments,compounds of the present disclosure are administered orally.

Kits

Provided herein are also kits that include a compound of the disclosure,or a pharmaceutically acceptable salt, tautomer, stereoisomer, mixtureof stereoisomers, prodrug, or deuterated analog thereof, and suitablepackaging. In one embodiment, a kit further includes instructions foruse. In one aspect, a kit includes a compound of the disclosure, or apharmaceutically acceptable salt, tautomer, stereoisomer, mixture ofstereoisomers, prodrug, or deuterated analog thereof, and a label and/orinstructions for use of the compounds in the treatment of theindications, including the diseases or conditions, described herein.

Provided herein are also articles of manufacture that include a compounddescribed herein or a pharmaceutically acceptable salt, tautomer,stereoisomer, mixture of stereoisomers, prodrug, or deuterated analogthereof in a suitable container. The container may be a vial, jar,ampoule, preloaded syringe, and intravenous bag.

Treatment Methods and Uses

“Treatment” or “treating” is an approach for obtaining beneficial ordesired results including clinical results. Beneficial or desiredclinical results may include one or more of the following: a) inhibitingthe disease or condition (e.g., decreasing one or more symptomsresulting from the disease or condition, and/or diminishing the extentof the disease or condition); b) slowing or arresting the development ofone or more clinical symptoms associated with the disease or condition(e.g., stabilizing the disease or condition, preventing or delaying theworsening or progression of the disease or condition, and/or preventingor delaying the spread (e.g., metastasis) of the disease or condition);and/or c) relieving the disease, that is, causing the regression ofclinical symptoms (e.g., ameliorating the disease state, providingpartial or total remission of the disease or condition, enhancing effectof another medication, delaying the progression of the disease,increasing the quality of life, and/or prolonging survival.

“Prevention” or “preventing” means any treatment of a disease orcondition that causes the clinical symptoms of the disease or conditionnot to develop. Compounds may, in some embodiments, be administered to asubject (including a human) who is at risk or has a family history ofthe disease or condition.

“Subject” refers to an animal, such as a mammal (including a human),that has been or will be the object of treatment, observation orexperiment. The methods described herein may be useful in human therapyand/or veterinary applications. In some embodiments, the subject is amammal. In one embodiment, the subject is a human.

The term “therapeutically effective amount” or “effective amount” of acompound described herein or a pharmaceutically acceptable salt,tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuteratedanalog thereof means an amount sufficient to effect treatment whenadministered to a subject, to provide a therapeutic benefit such asamelioration of symptoms or slowing of disease progression. For example,a therapeutically effective amount may be an amount sufficient todecrease a symptom of a disease or condition responsive to inhibition ofCot activity. The therapeutically effective amount may vary depending onthe subject, and disease or condition being treated, the weight and ageof the subject, the severity of the disease or condition, and the mannerof administering, which can readily be determined by one or ordinaryskill in the art.

The disclosure further relates to the use of said compounds for thetreatment and/or prophylaxis of diseases and/or conditions throughbinding of said nuclear receptor by said compounds. Further the presentdisclosure relates to the use of said compounds for the preparation of amedicament for the treatment and/or prophylaxis of diseases and/orconditions through binding of said nuclear receptor by said compounds.

Also provided herein are methods of treating a patient having a FXRmediated condition comprising administering a compound of formula (I),or pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a compound of formula (I), or pharmaceuticallyacceptable salt thereof.

In some embodiments, a compound of formula (I), or pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition comprising acompound of formula (I), or pharmaceutically acceptable salt thereof isprovided for use in the treatment of a FXR mediated condition.

In some embodiments, a compound of formula (I), or pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition comprising acompound of formula (I), or pharmaceutically acceptable salt thereof, isprovided for the manufacture of a medicament for the treatment of a FXRmediated condition.

In some embodiments, the FXR mediated condition is:

-   -   a chronic intrahepatic or some form of extrahepatic cholestatic        condition;    -   liver fibrosis;    -   an obstructive inflammatory disorder of the liver;    -   chronic inflammatory disorder of the liver;    -   liver cirrhosis;    -   liver steatosis or an associated syndrome;    -   cholestatic or fibrotic effects that are associated with        alcohol-induced cirrhosis or with viral-borne forms of        hepatitis;    -   liver failure or liver ischemia after major liver resection;    -   chemotherapy associated steatohepatitis (CASH);    -   acute liver failure; or    -   Inflammatory Bowel Disease.

In some embodiments, the FXR mediated condition is:

-   -   a lipid and lipoprotein disorder;    -   Type I Diabetes;    -   Type II Diabetes;        -   clinical complications of Type I and Type II Diabetes            selected from the group consisting of diabetic nephropathy,            diabetic neuropathy, diabetic retinopathy and other observed            effects of clinically manifest long term Diabetes;    -   Non-Alcoholic Fatty Liver Disease (NAFLD);    -   Non-Alcoholic Steatohepatitis (NASH);    -   obesity;    -   a metabolic syndrome selected from the group consisting of        combined conditions of dyslipidemia, diabetes and abnormally        high body-mass index;    -   acute myocardial infarction;    -   acute stroke; or    -   thrombosis which occurs as an endpoint of chronic obstructive        atherosclerosis.

In some embodiments, the FXR mediated condition is:

-   -   a non-malignant hyperproliferative disorder; and    -   a malignant hyperproliferative disorder selected from the group        consisting of hepatocellular carcinoma, colon adenoma, and        polyposis;    -   colon adenocarcinoma;    -   breast cancer;    -   pancreas adenocarcinoma;    -   Barrett's esophagus; or    -   other forms of neoplastic diseases of the gastrointestinal tract        and the liver.

In some embodiments, the FXR mediated condition is Non-AlcoholicSteatohepatitis (NASH).

In some embodiments, the present disclosure relates to the use ofcompounds according to Formula (I) in the preparation of a medicamentfor the prophylaxis and/or treatment of chronic intrahepatic or someforms of extrahepatic cholestatic conditions, of liver fibrosis, ofacute intraheptic cholestatic conditions, of obstructive or chronicinflammatory disorders that arise out of improper bile composition, ofgastrointestinal conditions with a reduced uptake of dietary fat andfat-soluble dietary vitamins, of inflammatory bowel diseases, of lipidand lipoprotein disorders, of Type II Diabetes and clinicalcomplications of Type I and Type II Diabetes, of conditions and diseaseswhich result from chronic fatty and fibrotic degeneration of organs dueto enforced lipid and specifically triglyceride accumulation andsubsequent activation of profibrotic pathways, of obesity and metabolicsyndrome (combined conditions of dyslipidemia, diabetes and abnormallyhigh body-mass index), of acute myocardial infarction, of acute stroke,of thrombosis which occurs as an endpoint of chronic obstructiveatherosclerosis, of persistant infections by intracellular bacteria orparasitic protozoae, of non-malignant hyperproliferative disorders, ofmalignant hyperproliferative disorders, of colon adenocarcinoma andhepatocellular carcinoma in particular, of liver steatosis andassociated syndromes, of liver failure or liver malfunction as anoutcome of chronic liver diseases or of surgical liver resection, ofHepatitis B infection, of Hepatitis C infection and/or of cholestaticand fibrotic effects that are associated with alcohol-induced cirrhosisor with viral-borne forms of hepatitis.

Medicaments as referred to herein may be prepared by conventionalprocesses, including the combination of a compound according to thepresent disclosure and a pharmaceutically acceptable carrier.

FXR is proposed to be a nuclear bile acid sensor. As a result, itmodulates both, the synthetic output of bile acids in the liver andtheir recycling in the intestine (by regulating bile acid bindingproteins). But beyond bile acid physiology, FXR seems to be involved inthe regulation of many diverse physiological processes which arerelevant in the etiology and for the treatment of diseases as diverse ascholesterol gallstones, metabolic disorders such as Type II Diabetes,dyslipidemias or obesity, chronic inflammatory diseases such asInflammatory Bowel Diseases or chronic intrahepatic forms of cholestasisand many other diseases.

FXR regulates a complex pattern of response genes in the liver and inthe gastrointestinal tract. The gene products have impact on diversephysiological processes. In the course of functional analysis of FXR,the first regulatory network that was analyzed was the regulation ofbile acid synthesis. While the LXRs induce the key enzyme of theconversion of cholesterol into bile acids, Cyp7A1, via the induction ofthe regulatory nuclear receptor LRH-1, FXR represses the induction ofCyp7A1 via the upregulation of mRNA encoding SHP, a further nuclearreceptor that is dominant repressive over LRH-1. Since FXR binds the endproducts of this pathway, primary bile acids such as cholic acid (CA) orCDCA, this can be regarded as an example of feedback inhibition on thegene expression level. Parallel to the repression of bile acid synthesisvia SHP, FXR induces a range of so-called ABC (for ATP-binding cassette)transporters that are responsible for the export of toxic bile acidsfrom the hepatocyte cytosol into the canaliculi, the small bile ductramifications where the bile originates. This hepatoprotective functionof FXR became first apparent with the analysis of FXR knockout mice.where under- or overexpression of several ABC-transporters in the liverwas shown. Further detailed analysis revealed that the major bile saltexcretory pump BSEP or ABCB11 (as well as the key enzyme which mediateslipid transfer from lipoproteins to phospholipids, PLTP, and the two keycanalicular membrane transporters for phospholipids, MRP-2 (ABCC4) andMDR-3 (ABCB4), are direct targets for ligand-directed transcriptionalactivation by FXR.

The fact that FXR seems to be the major metabolite sensor and regulatorfor the synthesis, export and re-circulation of bile acids suggested theuse of FXR ligands to induce bile flow and change bile acid compositiontowards more hydrophilic composition. With the development of the firstsynthetic FXR ligand GW4064 as a tool compound and of the semi-syntheticartificial bile acid ligand 6-alpha-ethyl-CDCA, the effects ofsuperstimulation of FXR by potent agonists could be analyzed. It wasshown that both ligands induce bile flow in bile duct ligated animals.Moreover, in addition to choleretic effects, also hepatoprotectiveeffects could be demonstrated. This hepatoprotective effect was furthernarrowed down to an anti-fibrotic effect that results from therepression of Tissue Inhibitors of Matrix-Metalloproteinases, TIMP-1 and2, the induction of collagen-deposit resolving Matrix-Metalloproteinase2 in hepatic stellate cells and the subsequent reduction ofalpha-collagen mRNA and Transforming growth factor beta (TGF-beta) mRNAwhich are both pro-fibrotic factors by FXR agonists. Furthermore,anti-cholestatic activity was demonstrated in bile-duct ligated animalmodels as well as in animal models of estrogen-induced cholestasis.

Genetic studies demonstrate that in hereditary forms of cholestasis(Progressive Familiar Intrahepatic Cholestasis=PFIC, Type I-IV) eithernuclear localization of FXR itself is reduced as a consequence of amutation in the FIC1 gene (in PFIC Type I, also called Byler's Disease)(F. Chen et al., Gastroenterology 2004, 126, 756; L. Alvarez et al.,Hum. Mol. Genet. 2004, 13, 2451) or levels of the FXR target geneencoding MDR-3 phospholipid export pump are reduced (in PFIC Type III).Taken together there is a growing body of evidence that FXR bindingcompounds will demonstrate substantial clinical utility in thetherapeutic regimen of chronic cholestatic conditions such as PrimaryBiliary Cirrhosis (PBC) or Primary Sclerosing Cholangitis (PSC).

The deep impact that FXR activation has on bile acid metabolism andexcretion is not only relevant for cholestatic syndromes but even moredirectly for a therapy against gallstone formation. Cholesterolgallstones form due to low solubility of cholesterol that is activelypumped out of the liver cell into the lumen of the canaliculi. It is therelative percentage of content of the three major components, bileacids, phospholipids and free cholesterol that determines the formationof mixed micelles and hence apparent solubility of free cholesterol inthe bile. FXR polymorphisms map as quantitative trait loci as one factorcontributing to gallstone disease. Using the synthetic FXR tool compoundGW4064 it could be demonstrated that activation of FXR leads to animprovement of the Cholesterol Saturation Index (CSI) and directly to anabolishment of gallstone formation in C57L gallstone susceptible micewhereas drug treatment in FXR knockout mice shows no effect on gallstoneformation.

These results qualify FXR as a good target for the development of smallmolecule agonists that can be used to prevent cholesterol gallstoneformation or to prevent re-formation of gallstones after surgicalremoval or shockwave lithotripsy.

Thus, in one embodiment of the disclosure, the compound according toFormula (I) and pharmaceutical compositions comprising said compound isused for the prophylaxis and/or treatment of obstructive or chronicinflammatory disorders that arise out of improper bile composition suchas cholelithiasis also known as cholesterol gallstones.

Beyond its strong hepatoprotective and choleretic as well asanti-fibrotic effects that FXR shows upon small molecule stimulatedactivation in the liver, FXR seems to have a role in protecting theintestine from neoplastic transformation and from the development ofpolyps and their transition into adenocarcinoma in the gut. Similar tothe situation in the intestine absence of FXR leads to a high increasein the formation of Hepatocellular Cacrcinoma (HCC), the most prominentform of liver cancer. Whereas a functional FXR prevents the formation ofcolon adenocarcinoma and hepatocellular carcinoma, FXR activationinduces liver regeneration after hepatectomy.

The combined hepatoprotective, anti-neoplastic and liver regenerativeeffects associated with FXR activation can be therapeutically exploitedfor the use of FXR agonists in the treatment of severe liver diseases.In one embodiment, the compounds according to the disclosure andpharmaceutical compositions comprising said compounds are used in thetreatment of liver diseases such as HCC, stimulation of liver regrowthand amelioration of side effects associated with major liver resection,liver cirrhosis independent of the etiology and prevention or treatmentof liver ischemia in the course of liver transplantation or major liversurgery.

Since the discovery of the first synthetic FXR agonist and itsadministration to rodents it became evident that FXR is a key regulatorof serum triglycerides. Over the past six years accumulating evidencehas been published that activation of FXR by synthetic agonists leads tosignificant reduction of serum triglycerides, mainly in the form ofreduced VLDL, but also to reduced total serum cholesterol.

But the lowering of serum triglycerides is not a stand alone effect.Treatment of db/db or ob/ob mice with synthetic FXR agonist GW4064resulted in marked and combined reduction of serum triglycerides, totalcholesterol, free fatty acids, ketone bodies such as 3-OH Butyrate.Moreover, FXR activation engages with the intracellular insulinsignaling pathway in hepatocytes, resulting in reduced output of glucosefrom liver gluconeogenesis but concomitant increase in liver glycogen.Insulin sensitivity as well as glucose tolerance were positivelyimpacted by FXR treatment. An effect on reduction of body weight wasalso recently observed in mice overfed with a high lipid diet. Thisweight loss effect might results from FXR's induction of FGF-19, afibroblast growth factor that is known to lead to weight loss andathletic phenotype. The effect of FXR agonist on reduction of bodyweight has been demonstrated.

Taken together, these pharmacological effects of FXR agonists can beexploited in different therapeutic ways: FXR binding compounds arethought to be good candidates for the treatment of Type II Diabetesbecause of their insulin sensitization, glycogenogenic, and lipidlowering effects.

In one embodiment, the compounds according to the disclosure andpharmaceutical compositions comprising said compounds are used in theprophylaxis and/or treatment of Type II Diabetes which can be overcomeby FXR-mediated upregulation of systemic insulin sensitivity andintracellular insulin signalling in liver, increased peripheral glucoseuptake and metabolisation, increased glycogen storage in liver,decreased output of glucose into serum from liver-borne gluconeogenesis.

In a further embodiment, said compounds and pharmaceutical compositionsare used for the prophylaxis and/or treatment of chronic intrahepatic,such as PBC, PSC, progressive familiar cholestasis (PFIC),alcohol-induced cirrhosis and associated cholestasis, and some forms ofextrahepatic cholestatic conditions, or liver fibrosis.

The disclosure also relates to a compound of Formula (I) or apharmaceutical composition comprising said compound for the prophylaxisand/or treatment of gastrointestinal conditions with a reduced uptake ofdietary fat and fat-soluble dietary vitamins which can be overcome byincreased intestinal levels of bile acids and phospholipids.

In a further embodiment, said compound or pharmaceutical composition isused for preventing and/or treating a disease selected from the groupconsisting of lipid and lipoprotein disorders such ashypercholesterolemia, hypertriglyceridemia, and atherosclerosis as aclinically manifest condition which can be ameliorated by FXR'sbeneficial effect on lowering total plasma cholesterol, lowering serumtriglycerides, increasing conversion of liver cholesterol into bileacids and increased clearance and metabolic conversion of VLDL and otherlipoproteins in the liver.

In one further embodiment, said compound and pharmaceutical compositionare used for the prophylaxis and/or treatment of diseases where thecombined lipid lowering, anti-cholestatic and anti-fibrotic effects ofFXR-targeted medicaments can be exploited for the treatment of liversteatosis and associated syndromes such as Non-Alcoholic Steatohepatitis(NASH), or for the treatment of cholestatic and fibrotic effects thatare associated with alcohol-induced cirrhosis, or with viral-borne formsof hepatitis.

In conjunction with the hypolipidemic effects it was also shown thatloss of functional FXR leads to increased atherosclerosis in ApoEknockout mice. Therefore, FXR agonists might have clinical utility asanti-atherosclerotic and cardioprotective drugs. The downregulation ofEndothelin-1 in Vascular Smooth Muscle Cells might also contribute tosuch beneficial therapeutic effects.

The disclosure also relates to a compound according to Formula (I) or apharmaceutical composition comprising said compound for preventive andposttraumatic treatment of a cardiovascular disorder, such as acutemyocardial infarction, acute stroke, or thrombosis which occur as anendpoint of chronic obstructive atherosclerosis.

Beyond controlling intestinal and colonic polyp formation, FXR seems tobe expressed in breast cancer tissue and cell lines but not in healthybreast tissue and seems to interact with the Estrogen Receptor in ERpositive breast cancer cells.

This would allow to regard FXR also as a potential target for thetreatment of proliferative diseases, especially metastasizing cancerforms that express a small molecule responsive form of FXR.

In a further embodiment, said compounds and pharmaceutical compositionsare used for the prophylaxis and/or treatment of malignanthyperproliferative disorders such as different forms of cancer,specifically certain forms of breast, liver or colon cancer whereinterference with an FXR ligand will have a beneficial impact.

Finally, FXR seems also to be involved in the control of antibacterialdefense in the intestine although an exact mechanism is not provided.From these published data, however, one can conclude that treatment withFXR agonists might have a beneficial impact in the therapy ofInflammatory Bowel Disorders (IBD), in particular those forms where theupper (ileal) part of the intestine is affected (e.g. ileal Crohn'sdisease) because this seems to be the site of action of FXR's control onbacterial growth. In IBD, the desensitization of the adaptive immuneresponse is somehow impaired in the intestinal immune system. Bacterialovergrowth might then be the causative trigger towards establishment ofa chronic inflammatory response. Hence, dampening of bacterial growth byFXR-borne mechanisms might be a key mechanism to prevent acuteinflammatory episodes.

Thus, the disclosure also relates to a compound according to Formula (I)or a pharmaceutical composition comprising said compound for preventingand/or treating a disease related to an Inflammatory Bowel Disease, suchas Crohn's disease or Colitis ulcerosa. FXR-mediated restoration ofintestinal barrier function and reduction in non-commensal bacterialload is believed to be helpful in reducing the exposure of bacterialantigens to the intestinal immune system and can therefore reduceinflammatory responses.

The disclosure further relates to a compound or pharmaceuticalcomposition for the prophylaxis and/or treatment of obesity andassociated disorders such as metabolic syndrome (combined conditions ofdyslipidemias, diabetes and abnormally high body-mass index) which canbe overcome by FXR-mediated lowering of serum triglycerides, bloodglucose and increased insulin sensitivity and FXR-mediated weight loss.

In a further embodiment, the compounds or pharmaceutical composition ofthe present disclosure are useful in preventing and/or treating clinicalcomplications of Type I and Type II Diabetes. Examples of suchcomplications include Diabetic Nephropathy, Diabetic Retinopathy,Diabetic Neuropathies, or Peripheral Arterial Occlusive Disease (PAOD).Other clinical complications of Diabetes are also encompassed by thepresent disclosure.

Furthermore, conditions and diseases which result from chronic fatty andfibrotic degeneration of organs due to enforced lipid and specificallytriglyceride accumulation and subsequent activation of profibroticpathways may also be prevented and/or treated by administering thecompounds or pharmaceutical composition of the present disclosure. Suchconditions and diseases encompass NASH and chronic cholestaticconditions in the liver, Glomerulosclerosis and Diabetic Nephropathy inthe kidney, Macula Degeneration and Diabetic Retinopathy in the eye andneurodegenerative diseases, such as Alzheimer's Disease in the brain, orDiabetic Neuropathies in the peripheral nervous system.

Dosage

The effective dosage of active ingredient employed may vary depending onthe particular compound employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Such dosage may be ascertained readily by a person skilled in the art.

When treating or preventing FXR mediated conditions for which compoundsof the present disclosure are indicated, generally satisfactory resultsare obtained when the compounds of the present disclosure areadministered at a daily dosage of from about 0.1 milligram to about 100milligram per kilogram of animal body weight. In some embodiments, thecompounds of the present disclosure are given as a single daily dose orin divided doses two to six times a day, or in sustained release form.For most large mammals, the total daily dosage is from about 1 milligramto about 1000 milligrams, or from about 1 milligram to about 50milligrams. In the case of a 70 kg adult human, the total daily dosewill generally be from about 7 milligrams to about 350 milligrams. Thisdosage regimen may be adjusted to provide the optimal therapeuticresponse. In some embodiments, the total daily dosage is from about 1milligram to about 900 milligrams, about 10 milligrams to about 800milligrams, about 20 milligrams to about 700 milligrams, about 30milligrams to about 600 milligrams, about 40 milligrams to about 550milligrams, or about 50 milligrams to about 400 milligrams.

The compounds of the present application or the compositions thereof maybe administered once, twice, three, or four times daily, using anysuitable mode described above. Also, administration or treatment withthe compounds may be continued for a number of days; for example,commonly treatment would continue for at least 7 days, 14 days, or 28days, for one cycle of treatment. Treatment cycles are well known incancer chemotherapy, and are frequently alternated with resting periodsof about 1 to 28 days, commonly about 7 days or about 14 days, betweencycles. The treatment cycles, in other embodiments, may also becontinuous.

In a particular embodiment, the methods provided herein compriseadministering to the subject an initial daily dose of about 1 to 800 mgof a compound described herein and increasing the dose by incrementsuntil clinical efficacy is achieved. Increments of about 5, 10, 25, 50,or 100 mg can be used to increase the dose. The dosage can be increaseddaily, every other day, twice per week, or once per week.

Combination Therapies

In some embodiments, a compound disclosed herein is administered incombination with one or more additional therapeutic agents to treat orprevent a disease or condition disclosed herein. In some embodiments,the one or more additional therapeutic agents are a(n) ACE inhibitor,Acetyl CoA carboxylase inhibitor, Adenosine A3 receptor agonist,Adiponectin receptor agonist, AKT protein kinase inhibitor,AMP-activated protein kinases (AMPK), Amylin receptor agonist,Angiotensin II AT-1 receptor antagonist, Autotaxin inhibitors, Bioactivelipid, Calcitonin agonist, Caspase inhibitor, Caspase-3 stimulator,Cathepsin inhibitor, Caveolin 1 inhibitor, CCR2 chemokine antagonist,CCR3 chemokine antagonist, CCRS chemokine antagonist, Chloride channelstimulator, CNR1 inhibitor, Cyclin D1 inhibitor, Cytochrome P450 7A1inhibitor, DGAT1/2 inhibitor, Dipeptidyl peptidase IV inhibitor,Endosialin modulator, Eotaxin ligand inhibitor, Extracellular matrixprotein modulator, Farnesoid X receptor agonist, Fatty acid synthaseinhibitors, FGF1 receptor agonist, Fibroblast growth factor (FGF-15,FGF-19, FGF-21) ligands, Galectin-3 inhibitor, Glucagon receptoragonist, Glucagon-like peptide 1 agonist, G-protein coupled bile acidreceptor 1 agonist, Hedgehog (Hh) modulator, Hepatitis C virus NS3protease inhibitor, Hepatocyte nuclear factor 4 alpha modulator (HNF4A),Hepatocyte growth factor modulator, HMG CoA reductase inhibitor, IL-10agonist, IL-17 antagonist, Ileal sodium bile acid cotransporterinhibitor, Insulin sensitizer, integrin modulator, intereukin-1receptor-associated kinase 4 (IRAK4) inhibitor, Jak2 tyrosine kinaseinhibitor, Klotho beta stimulator, 5-Lipoxygenase inhibitor, Lipoproteinlipase inhibitor, Liver X receptor, LPL gene stimulator,Lysophosphatidate-1 receptor antagonist, Lysyl oxidase homolog 2inhibitor, Matrix metalloproteinases (MMPs) inhibitor, MEKK-5 proteinkinase inhibitor, Membrane copper amine oxidase (VAP-1) inhibitor,Methionine aminopeptidase-2 inhibitor, Methyl CpG binding protein 2modulator, MicroRNA-21(miR-21) inhibitor, Mitochondrial uncoupler,Myelin basic protein stimulator, NACHT LRR PYD domain protein 3 (NLRP3)inhibitor, NAD-dependent deacetylase sirtuin stimulator, NADPH oxidaseinhibitor (NOX), Nicotinic acid receptor 1 agonist, P2Y13 purinoceptorstimulator, PDE 3 inhibitor, PDE 4 inhibitor, PDE 5 inhibitor, PDGFreceptor beta modulator, Phospholipase C inhibitor, PPAR alpha agonist,PPAR delta agonist, PPAR gamma agonist, PPAR gamma modulator,Protease-activated receptor-2 antagonist, Protein kinase modulator, Rhoassociated protein kinase inhibitor, Sodium glucose transporter-2inhibitor, SREBP transcription factor inhibitor, STAT-1 inhibitor,Stearoyl CoA desaturase-1 inhibitor, Suppressor of cytokine signalling-1stimulator, Suppressor of cytokine signalling-3 stimulator, Transforminggrowth factor β (TGF-β), Transforming growth factor β activated Kinase 1(TAK1), Thyroid hormone receptor beta agonist, TLR-4 antagonist,Transglutaminase inhibitor, Tyrosine kinase receptor modulator, GPCRmodulator, nuclear hormone receptor modulator, WNT modulators, orYAP/TAZ modulator.

Non-limiting examples of the one or more additional therapeutic agentsinclude:

ACE inhibitors, such as enalapril;

Acetyl CoA carboxylase (ACC) inhibitors, such as DRM-01, gemcabene,PF-05175157, and QLT-091382;

Adenosine receptor agonists, such as CF-102, CF-101, CF-502, andCGS21680;

Adiponectin receptor agonists, such as ADP-355;

Amylin/calcitonin receptor agonists, such as KBP-042;

AMP activated protein kinase stimulators, such as O-304;

Angiotensin II AT-1 receptor antagonists, such as irbesartan;

Autotaxin inhibitors, such as PAT-505, PAT-048, GLPG-1690, X-165,PF-8380, and AM-063;

Bioactive lipids, such as DS-102;

Cannabinoid receptor type 1 (CNR1) inhibitors, such as namacizumab andGWP-42004;

Caspase inhibitors, such as emricasan;

Pan cathepsin B inhibitors, such as VBY-376;

Pan cathepsin inhibitors, such as VBY-825;

CCR2/CCRS chemokine antagonists, such as cenicriviroc;

CCR2 chemokine antagonists, such as propagermanium;

CCR3 chemokine antagonists, such as bertilimumab;

Chloride channel stimulators, such as cobiprostone;

Diglyceride acyltransferase 2 (DGAT2) inhibitors, such as IONIS-DGAT2Rx;

Dipeptidyl peptidase IV inhibitors, such as linagliptin;

Eotaxin ligand inhibitors, such as bertilimumab;

Extracellular matrix protein modulators, such as CNX-024;

Farnesoid X receptor (FXR) agonists, such as AGN-242266, AKN-083,EDP-305, GNF-5120, LJN-452, LMB-763, obeticholic acid, Px-102, Px-103,M790, M780, M450, M480, PX20606, EYP-001, and INT-2228;

Farnesoid X receptor (FXR)/G-protein coupled bile acid receptor 1(TGR5)agonists, such as INT-767;

Fatty acid synthase inhibitors, such as TVB-2640;

Fibroblast growth factor 19 (rhFGF19)/cytochrome P450 (CYP)7A1inhibitors, such as NGM-282;

Fibroblast growth factor 21(FGF-21) ligand, such as BMS-986171,BMS-986036;

Fibroblast growth factor 21(FGF-21)/glucagon like peptide 1 (GLP-1)agonists, such as YH-25723;

Galectin-3 inhibitors, such as GR-MD-02;

Glucagon-like peptide 1(GLP1R) agonists, such as AC-3174, liraglutide,semaglutide;

G-protein coupled bile acid receptor 1(TGR5) agonists, such as RDX-009,INT-777;

Heat shock protein 47 (HSP47) inhibitors, such as ND-L02-s0201;

HMG CoA reductase inhibitors, such as atorvastatin, fluvastatin,pitavastatin, pravastatin, rosuvastatin, and simvastatin;

IL-10 agonists, such as peg-ilodecakin;

Ileal sodium bile acid cotransporter inhibitors, such as A-4250,volixibat potassium ethanolate hydrate (SHP-262), and GSK2330672;

Insulin sensitizers, such as, KBP-042, MSDC-0602K, Px-102, RG-125(AZD4076), and VVP-100X;

beta Klotho (KLB)-FGF1c agonist, such as NGM-313;

5-Lipoxygenase inhibitors, such as tipelukast (MN-001);

Lipoprotein lipase inhibitors, such as CAT-2003;

LPL gene stimulators, such as alipogene tiparvovec;

Liver X receptor (LXR) modulators, such as PX-L603, PX-L493, BMS-852927,T-0901317, GW-3965, and SR-9238;

Lysophosphatidate-1 receptor antagonists, such as BMT-053011, UD-009.AR-479, ITMN-10534, BMS-986020, and KI-16198;

Lysyl oxidase homolog 2 inhibitors, such as simtuzumab;

Semicarbazide-Sensitive Amine Oxidase/Vascular Adhesion Protein-1(SSAONAP-1) Inhibitors, such as PXS-4728A;

Methionine aminopeptidase-2 inhibitors, such as ZGN-839;

Methyl CpG binding protein 2 modulators, such as mercaptamine;

Mitochondrial uncouplers, such as 2,4-dinitrophenol;

Myelin basic protein stimulators, such as olesoxime;

NADPH oxidase 1/4 inhibitors, such as GKT-831;

Nicotinic acid receptor 1 agonists, such as ARI-3037M0;

NACHT LRR PYD domain protein 3 (NLRP3) inhibitors, such asKDDF-201406-03, and NBC-6;

Nuclear receptor modulators, such as DUR-928;

P2Y13 purinoceptor stimulators, such as CER-209;

PDE 3/4 inhibitors, such as tipelukast (MN-001);

PDE 5 inhibitors, such as sildenafil;

PDGF receptor beta modulators, such as BOT-191, BOT-509;

PPAR agonists, such as elafibranor (GFT-505), MBX-8025, deuteratedpioglitazone R-enantiomer, pioglitazone, DRX-065, saroglitazar, andIVA-337;

Protease-activated receptor-2 antagonists, such as PZ-235;

Protein kinase modulators, such as CNX-014;

Rho associated protein kinase (ROCK) inhibitors, such as KD-025;

Sodium glucose transporter-2(SGLT2) inhibitors, such as ipragliflozin,remogliflozin etabonate, ertugliflozin, dapagliflozin, andsotagliflozin;

SREBP transcription factor inhibitors, such as CAT-2003 and MDV-4463;

Stearoyl CoA desaturase-1 inhibitors, such as aramchol;

Thyroid hormone receptor beta agonists, such as MGL-3196, MGL-3745,VK-2809;

TLR-4 antagonists, such as JKB-121;

Tyrosine kinase receptor modulators, such as CNX-025;

GPCR modulators, such as CNX-023; and

Nuclear hormone receptor modulators, such as Px-102.

In certain specific embodiments, the one or more additional therapeuticagents are selected from A-4250, AC-3174, acetylsalicylic acid, AK-20,alipogene tiparvovec, aramchol, ARI-3037M0, ASP-8232, bertilimumab,Betaine anhydrous, BI-1467335, BMS-986036, BMS-986171, BMT-053011,BOT-191, BTT-1023, CAT-2003, cenicriviroc, CER-209, CF-102, CGS21680,CNX-014, CNX-023, CNX-024, CNX-025, cobiprostone, colesevelam,dapagliflozin, deuterated pioglitazone R-enantiomer, 2,4-dinitrophenol,DRX-065, DS-102, DUR-928, EDP-305, elafibranor (GFT-505), emricasan,enalapril, ertugliflozin, evogliptin, F-351, GKT-831, GNF-5120,GR-MD-02, hydrochlorothiazide, icosapent ethyl ester, IMM-124-E,INT-767, IONIS-DGAT2Rx, ipragliflozin, Irbesarta, propagermanium,IVA-337, JKB-121, KB-GE-001, KBP-042, KD-025, M790, M780, M450,metformin, sildenafil, LC-280126, linagliptin, liraglutide, LJN-452,LMB-763, MBX-8025, MDV-4463, mercaptamine, MGL-3196, MGL-3745,MSDC-0602K, namacizumab, NC-101, ND-L02-s0201, NGM-282, NGM-313,NGM-386, NGM-395, norursodeoxycholic acid, 0-304, obeticholic acid,25HC3S, olesoxime, PAT-505, PAT-048, peg-ilodecakin, pioglitazone,pirfenidone, PRI-724, PX20606, Px-102, PX-L603, PX-L493, PXS-4728A,PZ-235, RDX-009, remogliflozin etabonate, RG-125 (AZD4076),saroglitazar, semaglutide, simtuzumab, solithromycin, sotagliflozin,statins (atorvastatin, fluvastatin, pitavastatin, pravastatin,rosuvastatin, simvastatin), TCM-606F, TEV-45478, tipelukast (MN-001),TLY-012, TRX-318, TVB-2640, UD-009, ursodeoxycholic acid, VBY-376,VBY-825, VK-2809, vismodegib, volixibat potassium ethanolate hydrate(SHP-626), VVP-100X, WAV-301, WNT-974, and ZGN-839.

EXAMPLES

The following examples are included to demonstrate specific embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques to function well in the practice of the disclosure, and thuscan be considered to constitute specific modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the disclosure.

The compounds of the present disclosure can be prepared according to theprocedures of the following Schemes and Examples, using appropriatematerials and are further exemplified by the following specificexamples. Moreover, by utilizing the procedures described herein, inconjunction with ordinary skills in the art, additional compounds of thepresent disclosure claimed herein can be readily prepared. The compoundsillustrated in the examples are not, however, to be construed as formingthe only genus that is considered as the disclosure. The examplesfurther illustrate details for the preparation of the compounds of thepresent disclosure. Those skilled in the art will readily understandthat known variations of the conditions and processes of the followingpreparative procedures can be used to prepare these compounds. Forsynthesizing compounds which are embodiments described in the presentdisclosure, inspection of the structure of the compound to besynthesized will provide the identity of each substituent group. Theidentity of the final product will generally render apparent theidentity of the necessary starting materials by a simple process ofinspection, given the examples herein. The instant compounds aregenerally isolated in the form of their pharmaceutically acceptablesalts, such as those described above. In general, compounds describedherein are typically stable and isolatable at room temperature andpressure

The amine-free bases corresponding to the isolated salts can begenerated by neutralization with a suitable base, such as aqueous sodiumhydrogen carbonate, sodium carbonate, sodium hydroxide and potassiumhydroxide, and extraction of the liberated amine-free base into anorganic solvent, followed by evaporation. The amine-free base, isolatedin this manner, can be further converted into another pharmaceuticallyacceptable salt by dissolution in an organic solvent, followed byaddition of the appropriate acid and subsequent evaporation,precipitation or crystallization. The carboxylic free acidscorresponding to the isolated salts can be generated by neutralizationwith a suitable acid, such as aqueous hydrochloric acid, sodium hydrogensulfate, sodium dihydrogen phosphate, and extraction of the liberatedcarboxylic-free acid into an organic solvent, followed by evaporation.The carboxylic acid, isolated in this manner, can be further convertedinto another pharmaceutically acceptable salt by dissolution in anorganic solvent, followed by addition of the appropriate base andsubsequent evaporation, precipitation or crystallization.

An illustration of the preparation of compounds of the presentdisclosure is shown below. Unless otherwise indicated in the schemes,the variables have the same meaning as described above. The examplespresented below are intended to illustrate particular embodiments of thedisclosure. Suitable starting materials, building blocks and reagentsemployed in the synthesis as described below are commercially availablefrom Sigma-Aldrich or Acros Organics, for example, or can be routinelyprepared by procedures described in the literature, for example in“March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure”, 5^(th) Edition; John Wiley & Sons or T. Eicher, S. Hauptmann“The Chemistry of Heterocycles; Structures, Reactions, Synthesis andApplication”, 2′ edition, Wiley-VCH 2003; Fieser et al. “Fiesers'Reagents for organic Synthesis” John Wiley & Sons 2000.

General Synthetic Scheme

Compounds of Formula (I) wherein Y is N can be synthesized according tothe following general synthetic scheme.

In the general synthetic scheme above, X is a leaving group, PG is aprotecting group, and the remaining variables are as provided herein. Acompound of formula (C) can be prepared by reacting a compound offormula (A) with a compound of formula (B) in the presence of a base toform a compound of formula (C). A compound of formula (D) is formed froma compound of formula (C) under appropriate deprotection conditions. Acompound of formula (D) can be combined with a compound of formula (E)in the presence of a base to give a compound of Formula (I).

Appropriate compounds of structure (A) and (B) can be prepared accordingto the specific methods described in the following Examples or bymethods known in the art. In some embodiments, X is halo. In someembodiments, PG is BOC.

Step 1: 2-(3-hydroxyazetidin-1-yl)isonicotinonitrile (1a)

Potassium carbonate (4.6 g, 33 mmol) was added to2-chloro-4-pyridinecarbonitrile (2.0 g, 14.4 mmol) and3-hydroxyazetidine hydrochloride (1.7 g, 16 mmol) in NMP (12 mL) at roomtemperature, and the mixture was heated to 80° C. for 2 hrs in a sealedtube. The mixture was cooled to room temperature, treated with H₂O andextracted with EtOAc. The organic layers, were washed with brine, driedwith Na₂SO₄ and concentrated. Purification by chromatography (ISCO 24 gsilica column) using a gradient 1:1 hexanes/EtOAc—100% EtOAc gave2-(3-hydroxyazetidin-1-yl)isonicotinonitrile (1a).

Step 2: 2-(3-oxoazetidin-1-yl)isonicotinonitrile (1b)

N-methylmorpholine (1.9 g, 16 mmol) then tetrapropylammoniumperruthenate (190 mg, 0.5 mmol) were added to2-(3-hydroxyazetidin-1-yl)isonicotinonitrile (1.9 g, 10.7 mmol) inCH₂C₁₂ (200 mL) with molecular sieves (1 g, activated powdered, 4 Å) atroom temperature. After 20 minutes with vigorous stirring, the mixturewas filtered through a pad of Celite and concentrated. Purification bychromatography (ISCO 24 g silica column) using a gradient 100%hexanes—1:3 hexanes/EtOAc gave 2-(3-oxoazetidin-1-yl)isonicotinonitrile(1b).

Synthesis of 1c: (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane(1c)

To the solution of 4-bromo-3-chlorophenol (250 g, 1.21 mol) and TBSCl(272 g, 1.81 mol) in DMF (2.0 L) was added imidazole (164 g, 2.41 mol).Then the reaction was stirred at 30° C. for 12 h. The reaction mixturewas poured into H₂O (3 L) and extracted with EtOAc (2 L) twice. Thecombined organic layers were washed with H₂O (1 L) and brine (1 L),dried over Na₂SO₄, filtered and concentrated in vacuo. Purification bysilica gel chromatography eluted with petroleum ether gave(4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c).

Step 3:2-(3-(4-((tert-Butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(1d)

Isopropylmagnesium chloride lithium chloride complex (1.3 ml, 1.7 mmol,1.5 M in THF) was added dropwise to(4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (1c, 370 mg, 1.15mmol) in THF (0.9 ml) at room temperature. After 3 h, the reaction wascooled to 0° C. and treated with2-(3-oxoazetidin-1-yl)isonicotinonitrile (199 mg, 1.15 mmol) in oneportion as a solid. After 1 h, the reaction was quenched with H₂O andEtOAc. The organic layer was washed with brine, dried with Na₂SO₄ andconcentrated. Purification by chromatography (ISCO 4 g silica column)using a gradient 100% hexanes—1:3 hexanes/EtOAc gave2-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(1d).

Step 4:2-(3-(2-Chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(1e)

To a solution of2-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(1d) (180 mg, 0.43 mmol) in 2-MeTHF (4 mL) was added 1 M TBAF solutionin THF (0.6 mL, 0.59 mmol) at room temperature. After 30 minutes, themixture was quenched with water, extracted with EtOAc. The organic phasewas washed with brine (10 mL), dried with Na₂SO₄, and concentrate togive2-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(1e), which was used without further purification.

Step 1: 2,6-dichloro-4-fluorobenzaldehyde Oxime (2b)

A suspension of 2,6-dichloro-4-fluorobenzaldehyde (6.0 g, 31.2 mmol),NH₂OH.HCl (4.3 g, 62.4 mmol), Na₂CO₃ (8.3 g, 78.7 mmol) in ethanol-water(50 ml, 5:1) was stirred at room temperature for 3 h. The reaction wascondensed under vacuum and the residue was treated with water followedby extraction with ethyl acetate. The ethyl acetate layer was washedwith brine, dried over Na₂SO₄, and concentrated to afford2,6-dichloro-4-fluorobenzaldehyde oxime (2b).

Step 2: 2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl Chloride (2c)

To a solution of 2,6-dichloro-4-fluorobenzaldehyde oxime (2b, 5.5 g,26.7 mmol) in DMF (10 mL) was added N-chlorosuccinimide (4.3 g, 32.0mmol). The reaction was stirred at RT for 1 h. The mixture quenched withH₂O and extracted with EtOAc. The combined organic layers were washedwith brine, dried over anhydrous Na₂SO₄, filtered and concentrated togive the 2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl chloride (2c) thatwas used without further purification in the next step.

Step 3: Ethyl5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4-carboxylate(2d)

To a solution of 3-cyclopropyl-3-oxo-propionic acid ethyl ester (5.0 g,32.0 mmol) in 30 mL THF was added Et₃N (10.8 g, 107.2 mmol), thereaction was stirred at RT for 30 min, then the reaction mixture fromthe previous step (2,6-dichloro-4-fluoro-N-hydroxybenzimidoyl chloride(2c)) was added dropwise. The resulting mixture was stirred for 2 h atRT. The solvent was removed and the residue was partitioned with 100 mLwater and 50 mL EtOAc. The organic layer was washed with brine, dried,filtered, concentrated and purified by silica gel column (PE/EA=10/1) togive ethyl5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4-carboxylate(2d).

Step 4:(5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methanol(2e)

To the solution of ethyl5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole-4-carboxylate(2d, 3.4 g, 9.3 mmol) in THF (30 ml) was added LiAlH₄ (11.1 ml, 11.1mmol, 1 M in hexane) dropwise at 0° C. The reaction was stirred for 30min. 1.0 ml water was added, then 2.0 g 10% NaOH, 3.0 mL water wereadded. The mixture was filtered and concentrated. The crude was purifiedby silica gel column (PE/EA=2/1) to give(5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methanol(2e). LCMS (ESI): m/z 302.0 (M+1)⁺. ¹H NMR (500 MHz, CDCl₃): δ 7.22-7.20(d, J=8.5 Hz, 2H), 4.42-4.41 (d, J=6.0 Hz, 2H), 2.19-2.16 (m, 1H),1.41-1.39 (m, 1H), 1.29-1.26 (m, 2H), 1.16-1.13 (m, 2H).

Step 1: Methyl 5-fluoro-6-(3-hydroxyazetidin-1-yl)nicotinate (3a)

A mixture of azetidin-3-ol hydrochloride (2.8 g, 26 mmol), methyl6-bromo-5-fluoronicotinate (5.0 g, 21 mmol), and potassium carbonate(7.4 g, 53 mmol) in DMF (100 mL) was heated at 65° C. for 19 hours. Themixture was purified by flash chromatography (silica gel) to provide thedesired product. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₁₀H₁₂FN₂O3: 227.1;found: 227.0.

Step 2: methyl 5-fluoro-6-(3-oxoazetidin-1-yl)nicotinate (3b)

A solution of methyl 5-fluoro-6-(3-hydroxyazetidin-1-yl)nicotinate (4.7g, 21 mmol) in dichloromethane (270 mL) was treated with Dess-Martinperiodinane (9.7 g, 23 mmol). After 6 hours of stirring at roomtemperature, an additional portion of Dess-Martin periodinane (1.5 g)was added, and the mixture was allowed to stir overnight at roomtemperature. After stirring overnight, the mixture was treated withaqueous sodium thiosulfate solution and saturated aqueous sodiumhydrogen carbonate solution. The aqueous phase was extracted three timeswith dichloromethane. The combined extracts were dried over anhydrousmagnesium sulfate, filtered, concentrated to dryness under reducedpressure. The residue was purified twice by flash chromatography (silicagel) to provide the desired material. LCMS-ESI⁺ (m/z): [M+H₂O+H]⁺ calcdfor C₁₀H₁₂FN₂O₄: 243.1; found: 243.0.

Step 3: methyl6-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(3c)

A solution of (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane (4.5g, 14 mmol) in 2-methyltetrahydrofuran (14 mL) was treated withisopropylmagnesium chloride/lithium chloride solution (Aldrich, 1.3M, 11mL, 15 mmol) dropwise via syringe. The resulting mixture was stirred forapproximately one hour and then was cooled in an ice-water bath. Methyl5-fluoro-6-(3-oxoazetidin-1-yl)nicotinate (2.0 g, 8.9 mmol) was addedportions over 2 hours. The mixture was allowed to stand overnight atroom temperature. The mixture was quenched with 10% aqueous citric acidsolution. The aqueous phase was extracted three times with ethylacetate. The combined organics were washed once with saturated aqueoussodium chloride solution, dried over anhydrous magnesium sulfate,filtered, and concentrated under reduced pressure to provide the crudedesired product which was carried forward without further purification.LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₂₂H₂₉ClFN₂O₄Si: 467.2; found: 467.1.

Step 4: methyl6-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(3d)

Crude methyl6-(3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(approximately 10 mmol) was taken up in tetrahydrofuran (70 mL) andtreated with tetra-n-butylammonium fluoride solution (Aldrich, 1.0 M inTHF, 18 mL, 18 mmol). The mixture was allowed to stand at roomtemperature until deemed complete by LC/MS and then purified by flashchromatography (silica gel) to provide Intermediate 3d. LCMS-ESI⁺ (m/z):[M+H]⁺ calcd for C₁₆H₁₅ClFN₂O₄: 353.1; found: 353.0.

Example 1:5-((4-Bromo-3-chlorophenoxy)methyl)-4-cyclopropyl-1-(2,6-dichlorophenyl)-1H-pyrazoleStep 1: 2,4-difluorobenzaldehyde Oxime

This compound was synthesized according to the procedure as described inGeneral Synthesis 2, Step 1 starting with 2,4-difluorobenzaldehyde (10g, 70 mmol).

Step 2: 2,4-difluoro-N-hydroxybenzimidoyl Chloride

This compound was synthesized according to the procedure as described inGeneral Synthesis 2, Step 2 starting with 2,4-difluorobenzaldehyde oxime(9 g, 57 mmol).

Step 3: ethyl5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole-4-carboxylate

This compound was synthesized according to the procedure as described inGeneral Synthesis 2, Step 3 starting with2,4-difluoro-N-hydroxybenzimidoyl chloride (11 g, 57 mmol).

Step 4: (5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methanol

This compound was synthesized according to the procedure as described inGeneral Synthesis 2, Step 4 starting with ethyl5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole-4-carboxylate (2.2 g, 8mmol).

Step 5: 4-(Chloromethyl)-5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole

To a solution of(5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methanol (113 mg,0.45 mmol) in CH₂Cl₂ (2.3 mL) was added thionyl chloride (164 μL, 2.3mmol) at 0° C. The mixture was heated to reflux for 15 min and cooled toroom temperature. The mixture was concentrated in vacuo. AdditionalCH₂Cl₂ (5 mL) was added and the mixture was concentrated again. Thisprocess was repeated a third time to remove excess thionyl chloride. Thecrude residue was used in the next step without further purification.

Step 6:2-(3-(2-Chloro-4-((5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile

4-(chloromethyl)-5-cyclopropyl-3-(2,4-difluorophenyl)isoxazole (113 mg,0.45 mmol),2-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(Intermediate 1e) (149 mg, 0.5 mmol) and K₂CO₃ (124 mg, 0.9 mmol) werecombined in anhydrous DMF (2.3 mL) at room temperature. The mixture washeated to 65° C. under nitrogen. After 2 h, the solution was cooled toroom temperature, quenched with H₂O and extracted with EtOAc. Thecombined organic layers were washed with brine, dried over anhydrousNa₂SO₄, filtered and concentrated. Purification by chromatography: ISCO(12 g silica column) using a gradient of 100% CH₂Cl₂—3:1 CH₂Cl₂/premixed60:35:5 CH₂Cl₂:Et₂O:MeOH gave the title compound.

Step 7:2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinicAcid (Example 1)

10 M aqueous sodium hydroxide (0.67 ml) was added to2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinonitrile(210 mg, 0.39 mmol) in ethanol (2 mL) and H₂O (2 mL) at room temperatureand the mixture was heated at 60° C. for 90 minutes in a sealed tube.The mixture was cooled to room temperature and adjusted pH to about 5with 1 M HCl which caused a precipitate to fall out of solution. Thesolution was filtered and the solid was rinsed with Et₂O and dried invacuo to give2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,4-difluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinicacid (Example 1). ¹H NMR (300 MHz, DMSO-d6) ¹H NMR (300 MHz, DMSO-d₆) δ13.41 (s, 1H), 8.19 (dd, J=5.2, 0.8 Hz, 1H), 7.59 (td, J=8.5, 6.5 Hz,1H), 7.49-7.34 (m, 2H), 7.28-7.15 (m, 1H), 7.05-6.96 (m, 2H), 6.88-6.74(m, 2H), 6.20 (s, 1H), 5.00 (s, 2H), 4.47 (d, J=9.3 Hz, 2H), 4.18 (d,J=9.2 Hz, 2H), 2.40 (tt, J=8.3, 5.3 Hz, 1H), 1.20-1.00 (m, 4H). MS(ESI^(+) (m/z)) 554.0 (M+H).

Example 2:2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinicAcid

Synthesis of Intermediate A:

To a solution of (4-bromo-3-chlorophenoxy)(tert-butyl)dimethylsilane(1c, 60 g, 187 mmol) in THF (500 mL) was added dropwise n-BuLi (2.5 M,75 mL) at −78° C. under N2. The reaction was stirred at −78° C. for 1 h.Next a solution of tert-butyl 3-oxoazetidine-1-carboxylate (27 g, 155mmol) in THF (500 mL) was added dropwise to the mixture at −78° C. Thenthe reaction was stirred at 20° C. for 3 h. The reaction mixture waspoured into H₂O (1 L) and extracted with EtOAc (2 L) three times. Thecombined organic layers were washed with water (1 L), dried over Na₂SO₄,filtered and concentrated in vacuo. The crude product was purified bysilica gel chromatography eluted with 10:1 petroleum ether:EtOAc to give3-(4-((tert butyldimethylsilyl)oxy)-2-chlorophenyl)azetidin-3-ol(Intermediate A).

Step 1: tert-butyl3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidine-1-carboxylate

To a solution of tert-butyl3-(4-((tert-butyldimethylsilyl)oxy)-2-chlorophenyl)-3-hydroxyazetidine-1-carboxylate(Intermediate A, 1.27 g, 3.07 mmol) in THF (50.0 mL) at −10° C. wasadded 1M TBAF in THF (3.68 mL, 3.68 mmol) dropwise. The reaction wasstirred for 2 hours and was concentrated to afford tert-butyl3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidine-1-carboxylate, which wasused without further purification.

Step 2:4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole

A solution of(5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methanol(2e); 845 mg, 2.80 mmol) in DCM (28.0 mL) was cooled to 0° C. Thionylchloride (1.02 mL, 14.0 mmol) was added and the solution was heated at45° C. for 1 hour. The reaction was concentrated to dryness and usedwithout purification in the next step.

Step 3: tert-butyl3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate

A solution of tert-butyl3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidine-1-carboxylate (922 mg,3.07 mmol) in DMF (28.0 mL) was added to crude4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole,followed by the addition of potassium carbonate (773 mg, 5.60 mmol). Themixture was heated at 60° C. for 8 hours. The reaction was concentrated,diluted with water and extracted with EtOAc (3×). The combined organiclayers were washed with water, brine, dried over MgSO₄, filtered andconcentrated. The crude product was purified by silica gelchromatography (DCM/Et₂O/MeOH) to afford tert-butyl3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate.LCMS-ESI⁺ (m/z): [(M+H)—BOC]⁺ calcd 483.04; found 483.04.

Step 4:3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-ol

To a solution of tert-butyl3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidine-1-carboxylate(1.52 g, 2.60 mmol) in DCM (130 mL) was added 4 N HCl in 1,4-dioxane(26.0 mL, 104 mmol). The solution was stirred at room temperature for2.5 hours and was concentrated to dryness to afford3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-olas the hydrochloride salt, which was used without further purification.LCMS-ESr (m/z): [M+H]⁺ calcd 483.04; found 483.03.

Step 5: methyl2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinate

A mixture of methyl 2-bromopyridine-4-carboxylate (0.466 g, 2.16 mmol),3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-olas the hydrochloride salt (1.02 g, 1.96 mmol), cesium carbonate (2.56 g,7.85 mmol), (±)-BINAP (0.244 g, 0.392 mmol), palladium acetate trimer(88.0 mg, 0.131 mmol) and 1,4-dioxane (40.0 mL) was heated at 85° C. for18 hours. The reaction was cooled to room temperature, filtered overcelite and purified by silica gel chromatography (acetone/hexanes) toafford methyl2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinate.LCMS-ESI₊ (m/z): [M+H]⁺ calcd 618.08; found 618.20.

Step 6:2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinicAcid (Example 2)

To a solution of2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinate(617 mg, 0.997 mmol) in THF/water (1:1, 10 mL) was added lithiumhydroxide monohydrate (83.6 mg, 1.99 mmol). The solution was stirred for90 minutes, concentrated to remove THF and diluted with water. Aceticacid (0.23 mL, 3.99 mmol) was added while stirring which resulted in theprecipitation of solids. The solids were filtered, washed with water,IPA and ether, and dried under vacuum to afford2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinicacid (Example 2). LCMS-ESI⁺ (m/z): [M+H]⁺ calcd 604.06; found 604.15. ¹HNMR (300 MHz, DMSO-d₆) δ 13.47 (br s, 1H), 8.18 (dd, J=5.3, 0.8 Hz, 1H),7.69 (d, J=8.5 Hz, 2H), 7.37 (d, J=8.7 Hz, 1H), 7.02 (dd, J=5.3, 1.4 Hz,1H), 6.93 (d, J=2.6 Hz, 1H), 6.86 (br s, 1H), 6.75 (dd, J=8.6, 2.6 Hz,1H), 6.20 (s, 1H), 4.91 (s, 2H), 4.49 (d, J=9.3 Hz, 2H), 4.19 (d, J=9.3Hz, 2H), 2.46-2.37 (m, 1H), 1.23-1.04 (m, 4H).

Example 3:6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicAcid

Steps 1-4 were as described for the synthesis of Example 2.

Step 5: methyl6-(3-(2-chloro-4-((0-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate

A mixture of methyl 6-chloro-5-fluoropyridine (235 mg, 1.24 mmol),3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)azetidin-3-olas the hydrochloride salt (495 mg, 0.952 mmol) and potassium carbonate(1.05 g, 7.61 mmol) in DMF (30.0 mL) was heated at 60° C. for 1 hour.The reaction was concentrated, diluted with water and extracted withEtOAc (3×). The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated. The crude mixture was purified bysilica gel chromatography (DCM/Et₂O/MeOH) to afford methyl6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate.LCMS-ESI⁺ (m/z): [M+H]⁺ calcd 636.07; found 635.96.

Step 6:6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid (Example 3)

To a solution of methyl6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(364 mg, 0.571 mmol) in THF/water (1:1, 20.0 mL) was added lithiumhydroxide monohydrate (41.3 mg, 0.984 mmol). The solution was stirredfor 18 hours, concentrated to remove THF and diluted with water (10.0mL). The pH was adjusted to 3 using 1N HCl. The solids were filtered,washed with water, dissolved in ACN/water and lyophilized to afford6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid (Example 3). LCMS-ESI⁺ (m/z): [M+H]⁺ calcd 622.05; found 622.12. ¹HNMR (400 MHz, DMSO-d₆) δ 12.84 (bs, 1H), 8.44 (t, J=1.7 Hz, 1H),7.79-7.63 (m, 3H), 7.39 (d, J=8.7 Hz, 1H), 6.95 (d, J=2.5 Hz, 1H), 6.77(dd, J=8.6, 2.6 Hz, 1H), 6.28 (s, 1H), 4.93 (s, 2H), 4.70 (d, J=9.8 Hz,2H), 4.34 (d, J=9.5 Hz, 2H), 2.50-2.43 (m, 1H), 1.22-1.08 (m, 4H).

Intermediate 4:(3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methanol

Following General Synthesis 2, beginning with2,6-dichloro-4-fluorobenzaldehyde in Step 1 and using ethyl acetoacetatein Step 3,(3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methanol(Intermediate 4) was synthesized. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd 276.00;found 276.05.

Example 4: Preparation of6-(3-(2-chloro-4-((3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicAcid

Following the general procedure described for Example 3, usingintermediate 4,6-(3-(2-chloro-4-((3-(2,6-dichloro-4-fluorophenyl)-5-methylisoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid was synthesized. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd 596.04; found596.12. ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (bs, 1H), 8.44 (t, J=1.6 Hz,1H), 7.74-7.66 (m, 3H), 7.39 (d, J=8.7 Hz, 1H), 6.90 (d, J=2.6 Hz, 1H),6.75 (dd, J=8.7, 2.6 Hz, 1H), 6.26 (s, 1H), 4.87 (s, 2H), 4.69 (d, J=9.8Hz, 2H), 4.34 (d, J=9.8 Hz, 2H), 2.57 (s, 3H).

Example 5:6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicAcid

Step 1: (2,6-dichloro-4-fluorophenyl)hydrazine Hydrochloride

To a −5° C. solution (internal temperature, wet ice/acetone bath) of2,6-dichloro-4-fluoroaniline (3.0 g, 17 mmol) in 37% hydrochloric acid(30 mL) and trifluoroacetic acid (20 mL) was added dropwise an aqueoussolution of sodium nitrite (1.4 g, 20 mmol, 6 mL water). The reactionwas stirred for 90 minutes, then a solution of stannous chloridedihydrate (5.6 g, 25 mmol) in 37% hydrochloric acid (16 mL) was addedover 15 minutes, keeping the internal temperature ≤2° C. The mixture wasstirred overnight at room temperature. The mixture was filtered and thecollected solid was washed with isopropyl alcohol and dried under housevacuum to provide the title compound. LCMS-ESI+(m/z): [M+H]+ calcd forC₆H₆Cl₂FN₂: 195.0; found: 194.9.

Step 2: Ethyl4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazole-5-carboxylate

N,N-Dimethylformamide dimethyl acetal (2.7 mL, 20 mmol) was added toethyl 3-cyclopropyl-2-oxopropanoate (Synnovator, 1.6 g, 10 mmol) andstirred overnight at room temperature. The mixture was then concentratedto dryness under reduced pressure. To the residue was added successivelyethanol (40 mL), (2,6-dichloro-4-fluorophenyl)hydrazine hydrochloride(2.6 g, 11 mmol), and 37% hydrochloric acid (150 μL). The reaction wasstirred at room temperature for four hours, followed by 2 days ofheating at reflux. The cooled mixture was purified by flashchromatography (silica gel) to provide the title compound. LCMS-ESI+(m/z): [M+H]+ calcd for C₁₅H₁₄Cl₂FN₂O₂: 343.0; found: 343.1.

Step 3:(4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methanol

A solution of ethyl4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazole-5-carboxylate(1.5 g, 4.4 mmol) in tetrahydrofuran (50 mL) was cooled to between −12and −10° C. A solution of lithium aluminum hydride (Aldrich, 2 M intetrahydrofuran, 2.6 mL, 5.2 mmol) was added dropwise. The mixture wasallowed to stir for 35 minutes. The mixture was quenched (Fieserprocedure) and purified by flash chromatography (silica gel) to providethe title compound. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₁₃H₁₂Cl₂FN₂O:301.0; found: 301.1.

Step 4:5-(chloromethyl)-4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazole

Thionyl chloride (110 μL, 1.5 mmol) was added to a solution of(4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methanol(0.15 g, 0.51 mmol) in dichloromethane (2.5 mL). The mixture was heatedat 60° C. for 40 minutes and then concentrated under reduced pressure toprovide the crude desired product, which was carried forward withoutfurther purification. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₁₃H₁₁Cl₃FN2:319.0; found: 319.1.

Step 5: Methyl6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate

A solution of4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole(0.16 g, 0.51 mmol) in DMF (3 mL) was treated with methyl6-(3-(2-chloro-4-hydroxyphenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(0.20 g, 0.56 mmol), sodium iodide (0.13 g, 0.86 mmol), and potassiumcarbonate (0.14 g, 1.0 mmol). The mixture was heated 65° C. overnightand then purified by flash chromatography (silica gel) to provide thedesired material. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₂₉H₂₄Cl₃F₂N₄O₄:635.1; found: 635.2.

Step 6:6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicAcid (Example 5)

A mixture of methyl6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinate(0.35 g, 0.39 mmol) and lithium hydroxide monohydrate (49 mg, 1.2 mmol)were taken up in 1:1 aqueous tetrahydrofuran (6 mL), and the mixture wasstirred at room temperature. Upon completion, the mixture was acidifiedwith glacial acetic acid and concentrated. The residue was purified byflash chromatography (silica gel) to provide6-(3-(2-chloro-4-((4-cyclopropyl-1-(2,6-dichloro-4-fluorophenyl)-1H-pyrazol-5-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid (Example 5). LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₂₈H₂₂Cl₃F₂N₄O₄:621.1; found: 621.2. 1H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H), 8.44 (t,J=1.6 Hz, 1H), 7.76 (d, J=8.3 Hz, 2H), 7.70 (dd, J=12.7, 1.7 Hz, 1H),7.49 (s, 1H), 7.40 (d, J=8.7 Hz, 1H), 7.00 (d, J=2.6 Hz, 1H), 6.80 (dd,J=8.7, 2.6 Hz, 1H), 6.28 (s, 1H), 5.01 (s, 2H), 4.69 (d, J=9.8 Hz, 2H),4.34 (d, J=9.6 Hz, 2H), 1.89 (tt, J=8.4, 5.1 Hz, 1H), 0.93 (m, 2H), 0.65(m, 2H).

Example 6:5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-6-methoxynicotinicAcid

Synthesis of Intermediate 5 Step 1:(5-bromo-6-methoxypyridin-3-yl)methanol

To a solution of methyl 5-bromo-6-methoxynicotinate (52.8 g, 215.0 mmol)in THF (500 mL) was added DIBAL-H (1.0 M, in toluene) (344 ml, 344 mmol)at −20° C. Then the mixture was stirred at RT for 2 h. The mixture wasquenched with sat. NH₄Cl and diluted with ethyl acetate. The organicportion was washed with brine, dried over anhydrous sodium sulfate,filtered, concentrated under reduced pressure and purified by flashchromatography on silica gel (PE/EtOAc=4/1) to give the title compound.

Step 2:3-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridine

To a solution of (5-bromo-6-methoxypyridin-3-yl)methanol (42.2 g, 194mmol) and tert-butyldimethylsilyl chloride (35.0 g, 232 mmol) in CH₂Cl₂(500 ml) was added imidazole (19.8 g, 291 mmol). The mixture was stirredat RT for 8 h. The mixture was quenched with water and diluted withethyl acetate. The organic portion was washed with brine, dried overanhydrous sodium sulfate, filtered, concentrated under reduced pressureand purified by flash chromatography on silica gel (PE/EtOAc=10/1) togive the title compound.

Step 3:3-(benzyloxy)-1-(5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol

3-bromo-5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridine (61.2g, 184 mmol) was dissolved in absolute THF (500 mL) under argon) a 1.6 Msolution of n-butyllithium (138 mL, 221 mmol) in THF was added dropwiseat −78° C. The mixture was stirred for 30 min at the same temperature. Asolution of 3-(benzyloxy)cyclobutan-1-one (35.7 g, 202 mmol) in THF (100mL) was then added at −78° C., and the mixture was subsequently stirredat this temperature for 30 min. Saturated aqueous ammonium chloride wassubsequently added and the mixture was extracted with ethyl acetate. Theorganic phase was washed with water and saturated sodium chloridesolution, dried over magnesium sulfate and filtered. After removal ofthe solvent on a rotary evaporator, the residue was purified by flashchromatography on silica gel (PE/EtOAc=2/1) to give the title compound.

Step 4:3-(benzyloxy)-1-(5-(hydroxymethyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol

To a solution of3-(benzyloxy)-1-(5-(((tert-butyldimethylsilyl)oxy)methyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol(31.6 g, 73.6 mmol) in THF (300 mL) was added TBAF (88 mL, 1 mol/L). Themixture was stirred at rt for 6 hours, then poured into water andextracted with ethyl acetate. The organic phase was washed with waterand saturated sodium chloride solution, dried over magnesium sulfate andfiltered. The organic phase was concentrated to give the title compound.

Step 5: 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinic Acid

To a solution of3-(benzyloxy)-1-(5-(hydroxymethyl)-2-methoxypyridin-3-yl)cyclobutan-1-ol(23.2 g, 73.6 mmol) in MeCN (300 mL) and H₂O (100 mL) was addediodobenzene diacetate (64.4 g, 200 mmol) and TEMPO (7.86 g, 50 mmol),and the solution was stirred at room temperature for 2 hrs. The mixturewas quenched with sat. sodium bicarbonate solution and diluted withethyl acetate. The organic portion was washed with brine, dried overanhydrous sodium sulfate, filtered; the organic phase was concentratedto give the title compound.

Step 6: Methyl 5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinate

To a solution of5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinic acid (17.5 g,crude) in THF/MeOH (200/50 mL) was added TMSN₂CH₃ (50 mL, 20 mol/L) at0° C. The mixture was stirred at room temperature for 3 hours, thenpoured into water and extracted with ethyl acetate. The organic phasewas washed with water and saturated sodium chloride solution, dried overmagnesium sulfate and filtered. The filtrate was concentrated underreduced pressure and purified by flash chromatography on silica gel(PE/EA=10.1) to give the title compound.

Step 7: Methyl 5-(3-(benzyloxy)-1-fluorocyclobutyl)-6-methoxynicotinate

To a cooled solution of methyl5-(3-(benzyloxy)-1-hydroxycyclobutyl)-6-methoxynicotinate (15.2 g, 44.3mmol) in DCM (200 mL) was added DAST (8.0 mL) at −78° C. dropwise bysyringe. After stirring 5 minutes at −78° C., the reaction was allowedto warm to −20° C. and stirred for 75 minutes, then it was quenched withH₂O (100 mL), diluted with EtOAc and the phases were separated. Theorganic phase was washed with sat. aq. NaHCO₃ and brine, then dried overMgSO₄, filtered, and concentrated. The crude product was purified bychromatography (PE:EtOAc=4:1) to give the title compound.

Step 8: methyl 5-(3-hydroxycyclobutyl)-6-methoxynicotinate

To a solution of methyl5-(3-(benzyloxy)-1-fluorocyclobutyl)-6-methoxynicotinate (12.7 g, 3.68mmol) in MeOH (200 mL) and formic acid (10 mL) was added Pd black (3.0g). The reaction was stirred vigorously under N2. After about 1.5 hrs,additional Pd black was added (1.5 g) and the reaction stirredovernight. The reaction mixture was filtered and concentrated. Theresidue was dissolved in EtOAc and washed with sat. Na₂CO₃. The organicphase was dried over MgSO₄, filtered and concentrated to an oilyresidue. The residue was purified by chromatography (MeOH:CH₂Cl₂=1:20)to give the title compound.

Step 9: Methyl 6-methoxy-5-(3-oxocyclobutyl)nicotinate (Intermediate 5)

To a solution of methyl 5-(3-hydroxycyclobutyl)-6-methoxynicotinate (4.0g, 16.9 mmol) in MeCN (100 mL) and H₂O (30 mL) was added iodobenzenediacetate (16.1 g, 50 mmol) and TEMPO (2.92 g, 18.6 mmol), and thesolution was stirred at room temperature for 2 hrs. The mixture wasquenched with sat. Na₂CO₃ and then diluted with ethyl acetate. Theorganic portion was washed with brine, dried over anhydrous sodiumsulfate, filtered, and the organic phase was concentrated and purifiedby chromatography (PE:EA=5:1) to give Intermediate 5.

Step 10:4-((4-bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole

A solution of crude4-(chloromethyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole(prepared as described in Example 2, step 2; 0.42 g, 1.3 mmol) inN,N-dimethylformamide (DMF, 6 mL) was treated with4-bromo-3-chlorophenol (0.27 g, 1.3 mmol), sodium iodide (0.34 g, 2.2mmol), and potassium carbonate (0.37 g, 2.6 mmol). The mixture washeated at 60° C. for 35 minutes before it was cooled and purified byflash chromatography (silica gel) to provide the desired material.LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₁₉H₁₃BrCl₃FNO₂: 491.9; found: 492.0.

Step 11: Methyl5-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-6-methoxynicotinate

Under an atmosphere of Argon, a solution of4-(4-bromo-3-chlorophenoxy)methyl)-5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazole(0.83 g, 1.7 mmol) in 2-methyltetrahydrofuran (2 mL) was treated withisopropylmagnesium chloride/lithium chloride solution (Aldrich, 1.3 M intetrahydrofuran, 1.3 mL, 1.7 mmol) dropwise via syringe. After thepassage of four hours, an additional volume of isopropylmagnesiumchloride/lithium chloride solution (1.3 mL) was added. In a separatevessel, under an atmosphere of Argon, a solution of methyl6-methoxy-5-(3-oxocyclobutyl)nicotinate (Intermediate 5), 0.21 g, 0.90mmol) in tetrahydrofuran (5 mL) was treated with lanthanum (III)chloride/2 lithium chloride solution (Aldrich, 0.6 M in tetrahydrofuran,1.5 mL, 0.9 mmol). This mixture was stirred for one hour at roomtemperature before it was cooled in a −8° C. wet ice/acetone bath. TheGrignard solution from above was added dropwise to the ketone solutionvia syringe. The reaction mixture was stirred overnight under an Argonatmosphere. The mixture was quenched with saturated aqueous ammoniumchloride solution. The aqueous phase was extracted three times withethyl acetate. The combined organics were washed once with saturatedaqueous sodium chloride solution, dried over anhydrous magnesiumsulfate, filtered, and concentrated under reduced pressure. The cruderesidue was purified by flash chromatography (silica gel) to provide thetitle compound. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₃₁H₂₇Cl₃FN₂O₆: 647.1;found: 647.1.

Step 12:5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-6-methoxynicotinicAcid (Example 6)

A mixture of methyl5-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-6-methoxynicotinate(0.26 g, 0.40 mmol) and lithium hydroxide monohydrate (33 mg, 0.79 mmol)were taken up in 1:1 aqueous tetrahydrofuran (10 mL) and stirredovernight at room temperature. The volatiles were mostly removed byunder reduced pressure. The aqueous mixture was diluted with water andtreated dropwise with 10% aqueous hydrochloric acid. The resultingmixture was extracted with ethyl acetate three times. The combinedorganics were washed with saturated aqueous sodium chloride solution(with a small amount of hydrochloric acid added). The combined organicswere dried over anhydrous magnesium sulfate, filtered, and concentratedunder reduced pressure. The residue was purified first by flashchromatography (silica gel) and then by preparative HPLC(acetonitrile/water, TFA). The combined fractions collected by HPLC wereneutralized with saturated aqueous sodium hydrogen carbonate solution,saturated with sodium chloride, and extracted three times with ethylacetate. The combined organics were dried over anhydrous magnesiumsulfate, filtered, and concentrated. The residue was taken up in ethylacetate, treated with anhydrous magnesium sulfate, filtered, andconcentrated. Again the residue was taken up in ethyl acetate andfiltered through a pad of Celite diatomaceous earth. The filtrate wasconcentrated to provide5-((1S,3S)-3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxycyclobutyl)-6-methoxynicotinicacid (Example 6). LCMS-ESI⁺ r (m/z): [M+H]⁺ calcd for C₃₀H₂₅Cl₃FN₂O₆:633.1; found: 633.1. 1H NMR (400 MHz, DMSO-d6) δ 13.00 (bs, 1H), 8.58(d, J=2.2 Hz, 1H), 8.13 (dd, J=2.3, 0.8 Hz, 1H), 7.72 (d, J=8.5 Hz, 2H),7.51 (d, J=8.7 Hz, 1H), 6.94 (d, J=2.6 Hz, 1H), 6.79 (dd, J=8.7, 2.6 Hz,1H), 4.94 (s, 2H), 3.90 (s, 3H), 3.15-3.03 (m, 2H), 2.91 (p, J=8.8 Hz,1H), 2.49-2.41 (m, 1H), 2.41-2.30 (m, 2H), 1.21-1.09 (m, 4H).

Example 7:2-(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinamido)ethane-1-sulfonicAcid

A solution of6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid (Example 3, 0.11 g, 0.18 mmol) in DMF (4 mL) was treated with HATU(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, 0.10 g, 0.27 mmol) followed by taurine (34mg, 0.27 mmol) and N,N-diisopropylethylamine (90 μL, 0.54 mmol). Themixture was stirred overnight at room temperature and was then purifiedby preparative HPLC (water/acetonitrile/TFA). The combined fractionswere treated with ammonium hydroxide solution and concentrated to give2-(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinamido)ethane-1-sulfonicacid (Example 7) as the ammonium salt. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd forC₃₀H₂₆Cl₃F₂N₄O₇S: 729.1; found: 729.2. ¹H NMR (400 MHz, DMSO-d6) δ 8.34(m, 2H), 7.73-7.61 (m, 3H), 7.37 (d, J=8.6 Hz, 1H), 7.29-6.95 (m, 4H),6.92 (d, J=2.5 Hz, 1H), 6.75 (dd, J=8.6, 2.5 Hz, 1H), 6.22 (s, 1H), 4.90(s, 2H), 4.63 (d, J=9.6 Hz, 2H), 4.29 (d, J=9.6 Hz, 2H), 3.46 (q, J=6.5Hz, 2H), 2.63 (t, J=7.3 Hz, 2H), 2.45-2.38 (m, 1H), 1.16 (dt, J=8.5, 3.1Hz, 2H), 1.10 (dt, J=5.4, 2.9 Hz, 2H).

Example 8:(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycine

Step 1: Methyl(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycinate

A solution of6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinicacid (Example 3, 0.12 g, 0.19 mmol) in DMF (4 mL) was treated with HATU(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, 0.11 g, 0.29 mmol) followed by glycinemethyl ester hydrochloride (36 mg, 0.29 mmol) andN,N-diisopropylethylamine (100 μL, 0.58 mmol). The mixture was stirredovernight at room temperature and was then quenched with saturatedaqueous sodium hydrogen carbonate solution. The aqueous phase wasextracted twice with ethyl acetate. The combined extracts were washedonce with 1:1 saturated aqueous sodium chloride solution/saturatedaqueous sodium hydrogen carbonate solution, dried over anhydrousmagnesium sulfate, filtered, and concentrated under reduced pressure togive the desired product, which was carried forward without furtherpurification. LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₃₁H₂₆Cl₃F₂N₄O₆: 693.1;found: 693.2.

Step 2:(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycine(Example 8)

A mixture of crude methyl(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycinate(approximately 0.19 mmol) and lithium hydroxide monohydrate (38 mg, 0.91mmol) in aqueous tetrahydrofuran (2:1, 3 mL) was stirred at roomtemperature for 3.5 hours. The volatile solvent was removed underreduced pressure. The residue was diluted with water and acidified to pH1 with 10% aqueous hydrochloric acid. The acidic aqueous mixture wasextracted three times with ethyl acetate. The combined organic extractswere washed once with saturated aqueous sodium chloride solution, driedover anhydrous magnesium sulfate, filtered, and concentrated to drynessunder reduced pressure. The residue was purified by flash chromatography(silica gel) to provide(6-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichloro-4-fluorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)-5-fluoronicotinoyl)glycine(Example 8). LCMS-ESI⁺ (m/z): [M+H]⁺ calcd for C₃₀H₂₄Cl₃F₂N₄O₆: 679.1;found: 679.3. ¹H NMR (400 MHz, DMSO-d6) δ 12.68 (s, 1H), 8.68 (t, J=5.8Hz, 1H), 8.44 (t, J=1.7 Hz, 1H), 7.80 (dd, J=13.2, 1.8 Hz, 1H), 7.71 (d,J=8.5 Hz, 2H), 7.39 (d, J=8.7 Hz, 1H), 6.95 (d, J=2.5 Hz, 1H), 6.77 (dd,J=8.6, 2.6 Hz, 1H), 6.26 (s, 1H), 4.93 (s, 2H), 4.66 (d, J=9.5 Hz, 2H),4.32 (d, J=9.3 Hz, 2H), 3.87 (d, J=5.8 Hz, 2H), 2.48-2.42 (partiallyobscured by DMSO, m, 1H), 1.16 (m, 4H).

Example 9: FRET Activity Assay

Determination of a ligand mediated cofactor peptide interaction toquantify ligand binding to the nuclear receptor FXR was performed asfollows.

Preparation of human FXR alpha ligand binding domain: The human FXRalphaLBD was expressed in E. coli strain BL21(DE3) as an N-terminally GSTtagged fusion protein. The DNA encoding the FXR ligand binding domainwas cloned into vector pDEST15 (Invitrogen). Expression was undercontrol of an IPTG inducible T7 promoter. The amino acid boundaries ofthe ligand binding domain were amino acids 187-472 of Database entryNM_005123 (RefSeq). Expression and purification of the FXR-LBD: Anovernight preculture of a transformed E. coli strain was diluted 1:20 inLB-Ampicillin medium and grown at 30° C. to an optical density ofOD₆₀₀=0.4-0.6. Gene expression was then induced by addition of 0.5 mMIPTG. Cells were incubated an additional 6 h at 30° C., 180 rpm. Cellswere collected by centrifugation (7000×g, 7 min, rt). Per liter oforiginal cell culture, cells were resuspended in 10 mL lysis buffer (50mM Glucose, 50 mM Tris pH 7.9, 1 mM EDTA and 4 mg/mL lysozyme) and lefton ice for 30 min. Cells were then subjected to sonication and celldebris removed via centrifugation (22000×g, 30 min, 4° C.). Per 10 mL ofsupernatant 0.5 mL prewashed Glutathione 4B sepharose slurry (Qiagen)was added and the suspension kept slowly rotating for 1 h at 4° C.Glutathione 4B sepharose beads were pelleted by centrifugation (2000×g,15 sec, 4° C.) and washed twice in wash buffer (25 mM Tris, 50 mM KCl, 4mM MgCl₂ and 1M NaCl). The pellet was resuspended in 3 mL elution bufferper liter of original culture (elution buffer: 20 mM Tris, 60 mM KCl, 5mM MgCl₂ and 80 mM glutathione added immediately prior to use aspowder). The suspension was left rotating for 15 min at 4° C., the beadspelleted and eluted again with half the volume of elution buffer thanthe first time. The eluates were pooled and dialysed overnight in 20 mMHepes buffer (pH 7.5) containing 60 mM KCl, 5 mM MgCl₂ as well as 1 mMdithiothreitol and 10% (v/v) glycerol. The protein was analysed bySDS-Page.

The method measures the ability of putative ligands to modulate theinteraction between the purified bacterial expressed FXR ligand bindingdomain (LBD) and a synthetic biotinylated peptide based on residues676-700 of SRC-1 (LCD2, 676-700). The sequence of the peptide used wasB-CPSSHSSLTERHKILHRLLQEGSPS-COOH (SEQ ID NO: 1) where the N-terminus wasbiotinylated (B). The ligand binding domain (LBD) of FXR was expressedas fusion protein with GST in BL-21 cells using the vector pDEST15.Cells were lysed by sonication, and the fusion proteins purified overglutathione sepharose (Pharmacia) according to the manufacturersinstructions. For screening of compounds for their influence on theFXR-peptide interaction, the Perkin Elmer LANCE technology was applied.This method relies on the binding dependent energy transfer from a donorto an acceptor fluorophor attached to the binding partner of interest.For ease of handling and reduction of background from compoundfluorescence LANCE technology makes use of generic fluorophore labelsand time resolved detection Assays were done in a final volume of 25 μLin a 384 well plate, in a Tris-based buffer (20 mM Tris-HCl pH 7.5; 60mM KCl, 5 mM MgCl₂; 35 ng/μL BSA), containing 20-60 ng/wellrecombinantly expressed FXR-LBD fused to GST, 200-600 nM N-terminallybiotinylated peptide, representing SRC1 aminoacids 676-700, 200 ng/wellStreptavidin-xlAPC conjugate (Prozyme) and 6-10 ng/well Eu W1024-antiGST(Perkin Elmer). DMSO content of the samples was kept at 1%. Aftergeneration of the assay mix and diluting the potentially FXR modulatingligands, the assay was equilibrated for 1 h in the dark at rt inFIA-plates black 384 well (Greiner). The LANCE signal was detected by aPerkin Elmer VICTOR2VTM Multilabel Counter. The results were visualizedby plotting the ratio between the emitted light at 665 and 615 nm. Abasal level of FXR-peptide formation is observed in the absence of addedligand. Ligands that promote the complex formation induce aconcentration-dependent increase in time-resolved fluorescent signal.Compounds which bind equally well to both monomeric FXR and to theFXR-peptide complex would be expected to give no change in signal,whereas ligands which bind preferentially to the monomeric receptorwould be expected to induce a concentration-dependent decrease in theobserved signal.

To assess the agonistic potential of the compounds, EC₅₀ values weredetermined for example compounds and are listed below in Table 2 (FRETEC₅₀).

Example 10: Mammalian One Hybrid (M111) Assay

Determination of a ligand mediated Gal4 promoter driven transactivationto quantify ligand binding mediated activation of FXR was performed asfollows.

The cDNA part encoding the FXR ligand binding domain was cloned intovector pCMV-BD (Stratagene) as a fusion to the yeast GAL4 DNA bindingdomain under the control of the CMV promoter. The amino acid boundariesof the ligand binding domain were amino acids 187-472 of Database entryNM 005123 (RefSeq). The plasmid pFR-Luc (Stratagene) was used as thereporter plasmid, containing a synthetic promoter with five tandemrepeats of the yeast GAL4 binding sites, driving the expression of thePhotinus pyralis (American firefly) luciferase gene as the reportergene. In order to improve experimental accuracy the plasmid pRL-CMV(Promega) was cotransfected. pRL-CMV contains the constitutive CMVpromoter, controlling the expression of the Renilla reniformisluciferase. All Gal4 reporter gene assays were done in HEK293 cells(obtained from DSMZ, Braunschweig, Germany) grown in MEM withL-Glutamine and Earle's BSS supplemented with 10% fetal bovine serum,0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 100 unitsPenicilin/Streptavidin per mL at 37° C. in 5% CO₂. Medium andsupplements were obtained from Invitrogen. For the assay, 5×10⁵ cellswere plated per well in 96 well plates in 100 μL per well MEM withoutPhenol Red and L-Glutamine and with Earle's BSS supplemented with 10%charcoal/dextran treated FBS (HyClone, South Logan, Utah), 0.1 mMnonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, and 100units Penicilin/Streptavidin per mL, incubated at 37° C. in 5% CO₂. Thefollowing day the cells were >90% confluence. Medium was removed andcells were transiently transfected using 20 μL per well of anOptiMEM-polyethylene-imine-based transfection-reagent (OptiMEM,Invitrogen; Polyethyleneimine, Aldrich Cat No. 40,827-7) including thethree plasmids described above. MEM with the same composition as usedfor plating cells was added 2-4 h after addition of transfectionmixture. Then compound stocks, prediluted in MEM were added (finalvehicle concentration not exceeding 0.1%). Cells were incubated foradditional 16 h before firefly and renilla luciferase activities weremeasured sequentially in the same cell extract using aDual-Light-Luciferase-Assay system (Dyer et al., Anal. Biochem. 2000,282, 158-161). All experiments were done in triplicates.

To assess the FXR agonistic potency of the example compounds, potencywas determined in the M1H assay and is listed below in Table 2 (M1HEC₅₀).

TABLE 2 Example FRET EC₅₀ (nM) M1H EC₅₀ (nM) 1 263 3000 2 25 831 3 7.43.8 4 35 176 5 18 8.6 6 49 353 7 6.9 1696 8 8.1 1264

Example 11: Metabolite ID Assay in Human Liver Microsomes

The metabolic stability of Example 3 and Comparative Example 1 in humanliver microsomes was conducted according to the following procedure.Human liver microsomes (35 μL protein concentration 20 mg/mL), 350 μL of100 mM potassium phosphate buffer (pH 7.4), 245 μL of deionized waterand 0.7 μL of compound stock solution (5 mM) were combined in a 1.5 mLmicrocentrifuge tube. The tube was sealed and gently vortexed for 10seconds, then placed in an Eppendorf ThermoMixer C and pre-warmed at 37°C. with shaking at 1100 rpm for 5 minutes.

NADPH solution (70 μL; 10 mM in water) was added while shaking, themixture was aspirated several times with pipet, and 200 μL was removedto a fresh 1.5 mL microcentrifuge tube on ice containing 200 μL of coldacetonitrile. This aliquot was vortexed at high speed for 10 secondsthen placed on ice. After 30 and 60 minutes additional 200 μl aliquotswere removed and transferred to fresh 1.5 mL microcentrifuge tube on icecontaining 200 μL of cold acetonitrile. These were vortexed at highspeed for 10 seconds then placed on ice.

The chilled aliquots were centrifuged at 14,300 rpm in a microcentrifugefor 10 minutes at 10° C., then the supernatant was transferred to adeepwell (1 mL) 96 well plate and sealed with a silicon mat. The samplewas transferred to the Cool Stack of the autoinjector (temperature setto 10° C.) and 20 μL was injected into the Thermo Elite Orbitrap massspectrometer. 20 μL samples were analyzed by UPLC-MS in order toidentify and quantify the metabolites (Agilent 1290 G4220 binary pumpUPLC with Agilent G1316 TCC column oven; Waters Acquity UPLC BEH C18(130 Å pore size, 1.7 μm particle size, 2.1×50 mm) column held at 40°C.; Agilent 1290 G4212 DAD diode array with wavelength range 190 to 400nm; Thermo Electron Orbitrap Elite mass spectrometer in FTMS positivemode).

Final microsomal protein concentration: 1 mg/mL

Final NADPH concentration: 1 mM

Final substrate concentration: 5

Time points: 0, 30, 60 minutes

Incubation volume per time point: 200 μL

Comparative Example 1, a direct comparator to Example 3 that lacks a4-fluorophenyl substituent present in the compounds disclosed herein,was found to be metabolized to a diol compound (M1) under the conditionsdescribed above (Scheme 1). Incorporation of the 4-fluoro substituentinhibited formation of metabolite M1 under the same conditions.

Example 12: Assessment of In Vivo Pharmacodynamics in Cynomolgus Monkey

In vivo pharmacodynamics of a representative compound of Formula (I) anda comparative example compound were determined as follows.

Test Article and Formulation

Oral suspension doses of a representative compound of Formula (I)(Example 3) and Comparative Example 2 (Example 13/9 of U.S. Pat. No.9,139,539) were formulated at concentrations of 2, 6, 20, and 60 mg/mLin aqueous suspensions of 0.5% sodium carboxymethylcellulose (Na CMC),1% ethanol, and 98.5% 50 mM Tris buffer, at pH 8.

Animals

Each dosing group consisted of three male Cynomolgus monkeys. At dosing,the animals weighed between 2.5 and 4.4 kg.

Dosing

The test articles were administered to the monkeys via oral gavage at 5mL/kg. Prior to withdrawal, the gavage tube was flushed withapproximately 10 mL of water.

Sample Collection

Venous blood samples were taken at specified time points after dosingfrom each animal. The blood samples were collected and transferred intotubes containing potassium (K₂) EDTA anticoagulant.

Determination of FGF19 Concentrations in Plasma

The FGF19 ELISA assay kit from BioVendor (product number RD191107200R)was used to determine FGF19 concentrations in the collected bloodsamples.

Determination of Drug Concentration in Plasma

An aliquot of 50 μL of each plasma sample from the 10 and 30 mg/kgdosing groups and the t=0 samples from the 100 and 300 mg/kg groups weretreated with 200 μL of acetonitrile (ACN) containing internal standard.An aliquot of 25 μL of the remaining samples from the 100 mg/kg groupwas combined with 25 μL of blank plasma to effect a 1:2 dilution andtreated with 200 μL of acetonitrile (ACN) containing internal standard.An aliquot of 10 μL of the remaining samples from the 300 mg/kg groupwas combined with 40 μL of blank plasma to effect a 1:5 dilution andtreated with 200 μL of acetonitrile (ACN) containing internal standard.The above solutions were centrifuged at 5000 RPM for 10 minutes and 50μL of supernatant was transferred to a clean 96-well plate, followed bythe addition of 200 μL of water. An aliquot of 10 μL was injected to theAPI 5000 LC/MS/MS system. Samples exceeding the calibration range of theinstrument were diluted and re-analyzed.

HPLC Conditions

A Zorbax Extend C18 HPLC column (50×2.1 mm, 3.5μ) from AgilentTechnologies (Part #735700-902) was used. Mobile phase A contained anaqueous solution of 1% acetonitrile in 10 mM ammonium formate adjustedto pH 3.0 with formic acid. Mobile phase B contained and 10% 10 mMammonium formate in acetonitrile adjusted to pH 5.2 with formic acid. AThermo Aria multiplexer with two identical Agilent 1200 series binarypumps (P/N G1312A Bin Pump) was used for elution and separation. Theelution program used is set forth in the following Table 3.

TABLE 3 Mobile Mobile Time Flow Rate Phase A Phase B (sec) Step Comments(mL/min) (%) (%) 30 Sample Loading 0.50 85 15 180 Ramp 0.50 50 50 90Ramp 0.50 99 1 60 Elution 0.50 99 1 120 Re-equilibrium 0.50 85 15

An API 5000 triple quadrupole mass spectrometer from AB Sciex, FosterCity, Calif. was used in multiple reaction monitoring mode to quantifythe compounds. The mass spectrometry parameters used are set forth inthe following Table 4.

TABLE 4 Spray Collision Dryer Ion voltage Gas 1 Gas 2 gas temperaturesource (V) (Arb) (Arb) (Arb) (° C.) Turbo Ion 5500 70 50 6 550 Spray

Results

FGF19 levels were compared following oral administration of increasingdoses of Example 3 or Comparative Example 2 (3 to 300 mg/kg).Dose-dependent increases in plasma exposure were observed for bothcompounds and the maximal AUC achieved with each compound at 300 mg/kgwere comparable (FIG. 1). Example 3 dose-dependently increased plasmaFGF19, reaching a C_(max) of 16000 pg/ml at the highest dose (FIG. 2).Administration of Comparative Example 2 also caused increases in plasmaFGF19, but the maximal level of FGF19 was significantly lower (C_(max)3000 ng/ml) than for Example 3. Furthermore, maximal FGF19 induction byComparative Example 2 was achieved at 5 mg/kg; higher doses provided nofurther increase despite greater plasma drug exposures (FIG. 2). ThisExample demonstrates that IV or oral administration of Example 3 caninduce greater FGF19 levels than Comparative Example 2.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this disclosure. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the disclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

It is to be understood that while the disclosure has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of thedisclosure. Other aspects, advantages and modifications within the scopeof the disclosure will be apparent to those skilled in the art to whichthe disclosure pertains.

The invention claimed is:
 1. A compound of formula:

or a salt thereof.
 2. A compound of formula:


3. A compound of formula: